The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

DNA Repair 12 (2013) 899–911

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The excision of 3 penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair Chia-Chia Lee a , Ya-Chien Yang a,b , Steven D. Goodman c , Chien-Ju Lin a , Yi-An Chen a , Yi-ting Wang a , Wern-Cherng Cheng b , Liang-In Lin a,b , Woei-horng Fang a,b,∗ a

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, 100-02, Taiwan, ROC Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 100-02, Taiwan, ROC c Division of Biomedical Sciences, Herman Ostrow School of Dentistry of the University of Southern California, Los Angeles, CA 90089-0641, USA b

a r t i c l e

i n f o

Article history: Received 22 April 2013 Received in revised form 12 August 2013 Accepted 13 August 2013 Available online 5 September 2013 Keywords: Deoxyinosine Endonuclease V DNA polymerase I 3 -5 Exonuclease DNA mismatch 3 Penultimate site

a b s t r a c t Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3 -5 exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3 -penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3 penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3 -5 exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3 -penultimate deoxyinosines derived from endo V nicking reaction. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Deoxyinosine in DNA can arise from spontaneous deamination of a deoxyadenosine residue. Reactive oxygen species (ROS) from normal aerobic respiration as well as exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat can also enhance the formation of deoxyinosine [1,2]. Deoxyinosine in DNA is potentially mutagenic since it prefers to pair with dCTP during replication, yielding A:T to G:C transition mutations [3]. In Escherichia coli, repair of deoxyinosine is primarily performed by endonuclease Vmediated excision repair [4–7]. Although the base excision repair protein hypoxanthine-DNA glycosylase activities can be found in E. coli [8,9], when compared to endo V-mediated repair such activities were too small to be significantly detected both by in vivo analysis [5] and in vitro assay using crude extracts [6]. A genetic study of endo V deficient nfi mutant demonstrated over a 200-fold

∗ Corresponding author at: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002, Taiwan, ROC. Tel.: +886 2 23123456 ext 66926; fax: +886 2 23711574. E-mail address: [email protected] (W.-h. Fang). 1568-7864/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2013.08.003

increase in the frequency of mutations with HNO2 treatment, primarily A:T to G:C and G:C to A:T transitions as well as G:C to C:G transversions, however, the alkA mutant (deficiency in hypoxanthine glycosylase activity) did not significantly increase the mutagenic frequency under the same experimental conditions [10]. Endo V, encoded by nfi gene in E. coli, was first described by Gates and Linn, which is active upon DNA exposed to UV light, OsO4 , acids, or X-rays [11], and subsequently found to be active on AP sites, urea residues, mismatches, flap DNA, pseudo Y structures, loops, and hairpins [12,13]. Endo V is evolutionary conserved in living organisms; the human endo V was purified and characterized recently, which is most active on deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Expression of human endo V in E. coli cells deficient in nfi, mug and ung genes caused a 3-fold reduction in mutation frequency [14]. The repair mechanism of endo V-mediated pathway is not fully understood in E. coli and other organisms [7]. Endo V incises the DNA at the second phosphodiester bond 3 to the lesion, leaving a 3 hydroxyl and a 5 phosphate termini. The nfi homologue from Thermotoga maritima possesses a 3 -5 exonuclease activity that might be used for removal of damaged nucleotides, but similar exonuclease activities were not found in endo V from E. coli and mammalian cells. Therefore, additional enzymes are required to

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excise the deoxyinosine lesions in the endo V-mediated repair process as well as a polymerase to fill in the excised single-stranded gap. In our previous study, using a G-I substrate we found that pol I was required to repair deoxyinosine lesions and possibly involved in excision of deoxyinosine at 3 penultimate position of endo V incision site [6]. The pol I polypeptide has two functional domains: a large domain (Klenow fragment) that contains the polymerase and proofreading 3 -5 exonuclease, and a small fragment that contains a 5 -3 exonuclease activity [15]. Pol I is capable of correcting all possible mismatches at primer termini [16] while its proofreading activity is primarily required to maintain fidelity of lagging strand Okazaki fragment processing [17]. Although pol I is capable excising the wrongly incorporated nucleotides at a primer terminus as we have previously suggested [6], there is no report that the proofreading 3 -5 exonuclease of pol I plays an active role in any of the DNA repair pathways. Since the selectivity for 3 editing of pol I is attributed partially to the melting capacity of the DNA and partially to the polymerase binding site [18,19], promoting movement of the DNA into the exonuclease site by rejecting aberrant primer termini, we theorized that deoxyinosine at the 3 penultimate nucleotide of a nick would provoke melting as well as reduced fit for binding, just as a mismatch at the 3 penultimate site of a primer is a substrate for the 3 -5 exonuclease of pol I. Thus processing of 3 penultimate deoxyinosine could belong to its native capability of proofreading. Several studies [20,21] have indicated that mismatches embedded upstream of the primer terminus can be recognized by the proofreading-proficient DNA polymerase and ultimately can be excised by the 3 -5 exonuclease. However, it is also known that in the absence of a sufficient concentration of dNTPs, 3 -5 exonuclease activity of pol I will catalyze step wise degradation from a free 3 hydroxyl end of double-stranded homoduplex DNA in vitro [22]. Since almost all the DNA-pol I binding assays were performed in the absence of dNTPs [20,21], additional evidence is required to explain our previous observation of deoxyinosine processing [6]. To get more insight into endo V-mediated repair pathway, we added two potentially mutagenic deoxyinosine-containing heteroduplexes to the assay platform we previously established for endo V-mediated excision repair using a restriction endonuclease scoring system [6]. We also used extracts from a 3 -5 exonuclease deficient polA mutant to confirm the critical role of proofreading exonuclease in the endo V-mediated pathway. We found that deoxyinosine as well as all 12 possible mismatches at the 3 penultimate site of the primer can be excised via pol I’s proofreading function with similar efficiencies. These results broaden our knowledge of the proofreading capacity of pol I that not only recognizes and corrects mis-incorporation errors during DNA replication, but also actively use the 3 -5 exonuclease to excise damaged nucleotides in DNA repair. This study provides further support for the idea that 3 -5 exonuclease activity of E. coli pol I is the major exonuclease activity for removing 3 penultimate deoxyinosine of endo V nicking products. This piece of evidence could complete a multistep working model for endo V-mediate repair pathway.

2. Materials and methods 2.1. Materials E. coli strains NM522, RS5033 were as described [23]. Strains AB1157, JW5547 (nfi769::kan), and CM5409 (polA1 (Am)) were from E. coli Genetic Stock Culture Center, Yale University. Strains KA796 (polA+ ::Tn9) and KA796 mutant (polA-D424A::Tn9) were kindly provided by Dr. Laurent Jannière (Polskiej Akademii Nak). The chromosome polA region of these isogenic strains was replaced

with Tn9 cat-containing polA or polAexo (polA-D424A, 3 -5 exonuclease deficiency) [17]. Pol I (E. coli), Klenow fragment, Klenow fragment (3 -5 exo− ), E. coli DNA ligase, T4 polynucleotide kinase, soluble endo V (recombinant protein fusion with maltose binding protein), and restriction endonucleases were obtained from New England Biolabs. CpoI was purchased from Fermentas. RecBCD nuclease was purchased from EPICENTRE Biotechnologies. The unit definition and concentration of specific lot of enzymes were provided by manufacturers. 2.2. Bacteriophages for substrates preparation The G-I substrate was prepared using bacteriophage M13LR1 as described [6]. The G-I substrate (Table 1) was dubbed dI-G previously [6]. We changed the name to accommodate the correct nucleotide in front of the misincorporated nucleotide. The M13WX1 was constructed for T-I preparation by inserting a 26-bp synthetic linker into unique XbaI site of phage M13mp18 (Fig. 1A and Table 1). The f1PMA was constructed for A-I preparation by inserting a 29-bp synthetic linker into the XbaI site of phage f1PM [24] (Fig. 1B and Table 1). A series of insertion mutants (i.e. f1PMA, f1PMC, f1PMG, and f1PMT in Fig. 1B and Table 2) were selected during our cloning process since we used a degenerate strategy at a specific site (Fig. 1B). Each insertion derivative contains a new, unique restriction endonuclease recognition sequence for identification and the insertions were confirmed by sequencing. Pairing different f1PM derivatives therefore generates all possible mismatches (Table 2), and thus could be used for making substrates containing 3 penultimate mismatch for the pol I proofreading assay (Fig. 1B and Table 2). 2.3. Construction of deoxyinosine-containing DNA substrates The deoxyinosine containing substrates were prepared by ligation of synthetic linker to gapped duplex DNA as described previously [6]. Taking G-I as an example, the M13mp18 replicative form DNA was digested with HindIII and mixed with a 4-fold molar excess of M13LR1 viral DNA, followed by alkaline denaturation and re-annealing. The excess single-stranded viral DNA was removed by hydroxyapatite (Biorad) chromatography and benzoylated naphthylated DEAE cellulose (Sigma) chromatography, and the linear double strand DNA was removed by RecBCD nuclease (EPICENTRE) digestion. After phenol extraction, the resulting circular duplex DNA containing 22-nt gap was purified by Vivaspin 20 ultrafiltration (GE Healthcare). A 5 -phosphorylated deoxyinosinecontaining 22-mer oligonucleotides (Blossom Biotech, Table 1) was then annealed to the gap and sealed by T4 DNA ligase in the presence of ethidium bromide [23]. The covalently closed G-I heteroduplex DNA was isolated by CsCl/ethidium bromide density gradient centrifugation. The T-I and A-I DNA substrates were prepared using a similar protocol with respective phage vectors and deoxyinosine-containing oligonucleotides as shown in Table 1. The covalently-closed-circular deoxyinosine containing substrates were subjected to endo V nicking assay to check their reactivity. A 10 ␮l nicking reaction included 0.15 U endo V, 0.1 ␮g (21 fmol) heteroduplex substrate, 50 mM potassium acetate, 1 mM dithiothreitol, 10 mM magnesium acetate and 20 mM Tris–acetate (pH7.9). After incubation at 37 ◦ C for specified times the reaction were stopped by heating at 75 ◦ C for 20 min. The nicking products were detected by agarose gel electrophoresis and ethidium bromide staining; and quantified using a CCD camera and NIH Image J 1.45s software. Substrates containing a deoxyinosine at 3 penultimate of a nick were prepared by digesting 10 ␮g covalently-closed-circular A-I, G-I, and T-I substrates with endo V at 37◦ C for 50 min in a reaction buffer as described above. The nicking product was purified

C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

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Table 1 Deoxyinosine substrates. Deoxyinosine substrate A-I G-I T-I

Phage

Repaired products

Scoring marker

V 5 -CGGTCCATAAGGTGG

f1PMA

CGGTCCATAAGGTGG

PflMI

C 3 -GCCAGGIATTCCACC

Linker

GCCAGGTATTCCACC

V 5 -AGCCTCGAGAGCTTG

M13LR1

AGCCTCGAGAGCTTG

C 3 -TCGGAGITCTCGAAC

Linker

TCGGAGCTCTCGAAC

V 5 -CCATGGTGTGGATGG

M13WX1

CCATGGTGTGGATGG

C 3 -GGTACCICACCTACC

Linker

GGTACCACACCTACC

XhoI XcmI

Covalently closed circular heteroduplexes containing a deoxyinosine lesion (‘I’) were prepared using the bacteriophage DNA shown as vectors. Synthetic oligonucleotides containing deoxyinosine were used to complement gapped duplex for substrate preparation. In the presence of deoxyinosine, the substrates were refractory to the restriction endonuclease scoring. After the repair, DNA products become sensitive to restriction endonuclease cleavage. The recognition sequences of corresponding restriction endonuclease markers for repair products are shown with underlines.

by phenol extraction and ethanol precipitation then quantified as described above. The nicking efficiencies of all three dI-containing substrates were over 97%. 2.4. Preparation of heteroduplex containing a single mismatch at 3 penultimate of a nick A heteroduplex containing 1 of 12 possible mismatches can be constructed by pairing different f1PM derivatives (Table 2). Using the restriction endonuclease BsaI to cleave the replicative form of the f1PM mutant will generate the strand break serving as 3 end of primer in the complementary strand of the final heteroduplex substrates (Table 2). Preparation of the replicative form double stranded DNAs and single-stranded bacteriophage DNAs were as described [23]. Heteroduplex DNA substrates were constructed as described [23] with minor modifications. In brief, phage f1PM derivative RF DNA (1 mg) was linearized with BsaI and mixed with a 4-fold molar excess of viral DNA, followed by alkaline denaturation and annealing. After isolation by hydroxyapatite chromatography, double strand DNA was dialyzed and then linear homoduplex DNA was removed by RecBCD DNase hydrolysis.

The open circular heteroduplex was purified by Vivaspin-20 (GE-Healthcare) dialysis and benzoylated naphthylated DEAE cellulose (Sigma) chromatography. Following extensive dialysis the heteroduplex was purified to near homogeneity. Linear form A-A and C-A substrates were prepared by digesting circular heteroduplex with AlwNI. The linear heteroduplex was then purified by phenol extraction and ethanol precipitation. A gapped G-G heteroduplex was prepared by digesting RF DNA of f1PMC with both BsaI and Hind III, and then pairing with single-stranded f1PMG viral DNA. The resulting G-G heteroduplex contains 24-nt gap 3 to the penultimate mismatch. 2.5. Repair assay using cell-free extracts E. coli cell-free extracts were prepared as described [23]. Repair assays for cell-free extracts were as described [6]. Briefly, DNA substrate (0.1 ␮g, 21 fmol) was incubated with 90 ␮g of E. coli extracts in 20-␮l reactions containing 20 mM Tris–HCl (pH 7.6), 50 ␮g/ml bovine serum albumin, 5 mM MgCl2 , 1 mM ATP, and 0.1 mM each of the four dNTPs. Reactions were incubated for 30 min at 37 ◦ C and stopped by adding 40 ␮l of 40 mM EDTA (pH 8.0). DNA was

Table 2 Substrates containing 3 penultimate mismatches for proofreading. Heteroduplex substrate A-A A-C A-G C-A C-C C-T G-A G-G G-T T-C T-G T-T

Phages

Corrected products

Scoring markers

V 5 -CGGTCCATAAGGTGG

f1PMA

CGGTCCATAAGGTGG

PflMI

C 3 -GCCAGGAATTCCACC

f1PMT

GCCAGGTATTCCACC

V 5 -CGGTCCATAAGGTGG

f1PMA

CGGTCCATAAGGTGG

C 3 -GCCAGGCATTCCACC

f1PMG

GCCAGGTATTCCACC

V 5 -CGGTCCATAAGGTGG

f1PMA

CGGTCCATAAGGTGG

C 3 -GCCAGGGATTCCACC

f1PMC

GCCAGGTATTCCACC

V 5 -CGGTCCCTAAGGTGG

f1PMC

CGGTCCCTAAGGTGG

C 3 -GCCAGGAATTCCACC

f1PMT

GCCAGGGATTCCACC

V 5 -CGGTCCCTAAGGTGG

f1PMC

CGGTCCCTAAGGTGG

C 3 -GCCAGGCATTCCACC

f1PMG

GCCAGGGATTCCACC

V 5 -CGGTCCCTAAGGTGG

f1PMC

CGGTCCCTAAGGTGG

C 3 -GCCAGGTATTCCACC

f1PMA

GCCAGGGATTCCACC

V 5 -CGGTCCGTAAGGTGG

f1PMG

CGGTCCGTAAGGTGG

C 3 -GCCAGGAATTCCACC

f1PMT

GCCAGGCATTCCACC

V 5 -CGGTCCGTAAGGTGG

f1PMG

CGGTCCGTAAGGTGG

C 3 -GCCAGGGATTCCACC

f1PMC

GCCAGGCATTCCACC

V 5 -CGGTCCGTAAGGTGG

f1PMG

CGGTCCGTAAGGTGG

C 3 -GCCAGGTATTCCACC

f1PMA

GCCAGGCATTCCACC

V 5 -CGGTCCTTAAGGTGG

f1PMT

CGGTCCTTAAGGTGG

C 3 -GCCAGGCATTCCACC

f1PMG

GCCAGGAATTCCACC

V 5 -CGGTCCTTAAGGTGG

f1PMT

CGGTCCTTAAGGTGG

C 3 -GCCAGGGATTCCACC

f1PMC

GCCAGGAATTCCACC

V 5 -CGGTCCTTAAGGTGG

f1PMT

CGGTCCTTAAGGTGG

C 3 -GCCAGGTATTCCACC

f1PMA

GCCAGGAATTCCACC

PflMI PflMI Bsu36I Bsu36I Bsu36I CpoI CpoI CpoI AflII AflII AflII

Circular heteroduplexes containing a set of base pair mismatches were prepared using the phage DNAs shown. With the exception of the mismatches (bold type), all heteroduplexes were otherwise identical. The local sequence environment allowed placement of mispairs within overlapping restriction sites that served as proofreading markers (underline). Complementary and viral strands are designated as C and V. A BsaI cleavage site was designed so that during substrate preparation it can provide a nick at the second phosphodiester bond 3 to the mismatches on the C strands (between GG residues of fifth and sixth positions from 3 end).

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A

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▼

V 5’-CTAGTCCATACCATGGTGTGTATGGA C 3'AGGTATGGTACCICACCTACC TGATC ▲ XcmI 3'

5'

XbaI 6257

5'

V

C

2.6. Reconstituted endo V-mediated excision repair

0/7277

3' M13mp18 AlwNI 2186

B

The resulting products were separated by agarose gel electrophoresis, and detected by ethidium bromide staining. The gel-images were captured by a gel documentation CCD camera (UVP Ltd.) using Viewfinder 3.0, and band intensities were then measured by NIH Image J 1.45s software.

▼

f1PMA 5'.......CCATAAGGTGG..3' PflMI 3'.......GGTATTCCACC..5' ▲

▼

f1PMC 5'........CCTAAGG.....3' Bsu36I 3'........GGATTCC.....5' ▲ ▼

f1PMG 5'...CGGACCG..........3' CpoI 3'...GCCTGGC..........5' ▲

▼

f1PMT 5'........CTTAAG......3' AflII 3'........GAATTC......5' ▲ ▼

V 5'-CTAGGGTCTCCGGACCXTAAGGTGGCA C 3'CCAGAGGCCTGG YATTCCACCGTGATC ▲ BsaI

Repair reactions with purified proteins were modified from our previous report [6]. In a 10 ␮l complete reaction, the enzyme mixture included 2.1 nM DNA substrate, 1.6 nM endo V, 20 nM pol I or its variants, and 2 U DNA ligase. The reaction buffer contained 10 mM Tris–HCl (pH 7.9), 10 mM magnesium chloride, 50 mM sodium chloride, 1 mM dithiothreitol, 50 ␮g/ml bovine serum albumin, 0.3 mM NAD− , and 125 ␮M each of the four dNTPs. The incubation was at 37 ◦ C for 30 min and the reactions were terminated by heat inactivation at 75 ◦ C for 20 min. The repair product was then analyzed by restriction endonuclease digestion and agarose gel electrophoresis as described above. 2.7. Correction of 3 penultimate mismatches by pol I These reactions were performed using 0.1 ␮g (23 fmol) heteroduplex substrate and different concentration of pol I in 10 ␮l reaction with 1 mM of each dATP, dCTP, dGTP and dTTP, 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2 , and 10 mM Tris–HCl (pH 7.9). After incubation at 37 ◦ C for specified times the reaction were stopped by heating at 75 ◦ C for 20 min. The correction product was treated with 1 U AlwNI in combination with 1 U indicated diagnostic restriction enzymes listed in Table 2 in manufacturer recommended buffers at 37 ◦ C for 60 min. The DNA products were analyzed, and quantified as described in Section 2.5. 3. Results 3.1. Construction of T-I and A-I substrates

3' V

5' C

5'

XbaI HindIII 5839 5815

0/6674

f1PM

3' AlwNI 2192

Fig. 1. Maps of M13mp18 and f1PM. (A) The map of bacteriophage M13mp18 replicative form DNA shows restriction enzyme sites relevant to this study. The synthetic oligodeoxyribonucleotides shown was inserted into the XbaI site. The resulting mutant M13WX1 contains an XcmI recognition sequence (bold). The deoxyinosine lesion (‘I’) was designed adjacent to the cleavage site of XcmI (solid triangles). (B) The map of bacteriophage f1PM replicative form DNA shows restriction enzyme sites relevant to this study. The positions of ‘X’ and ‘Y’ in the synthetic linker were randomized with all four possible nucleotides (underlines in resulting mutants). Each of the f1PM mutants can be identified by a unique restriction endonuclease (recognition sequences in bold and solid triangles pointing to the cleavage sites). The insertion derivatives of bacteriophage f1PM were used to construct 3 penultimate mismatch substrates. A BsaI site (bold) was designed in the duplex which can be cleaved at the second phosphodiester bond 3 to the mismatches of the complementary strands (solid triangle pointing to the C strand). A synthetic oligodeoxyribonucleotide with a deoxyinosine at ‘Y’ position was used for A-I substrate preparation.

isolated by phenol extraction and ethanol precipitation, and was then digested with 1 U AlwNI and the indicated scoring restriction enzymes (i.e. 1 U PflM1 for A-I, 1 U XhoI for G-I and 0.1 U XcmI for T-I) in manufacturer recommended buffers at 37 ◦ C for 60 min.

In our previous study, a deoxyinosine (dI) containing G-I heteroduplex DNA was constructed, in which the deoxyinosine resided in a disrupted XhoI recognition site of bacteriophage M13mp18 DNA [6]. We utilized a restriction endonuclease assay to score for the repair of G-I substrate. In the presence of deoxyinosine lesion, heteroduplex DNA is refractory to restriction endonuclease cleavage. After in vitro repair, the recognition sequence of XhoI was restored and repair level can be scored by the extent of restriction digestion [6]. We demonstrated that this assay platform is very effective for evaluating deoxyinosine repair efficiency both in crude cell extracts and purified proteins. However, the majority of deoxyinosine formed in DNA is through spontaneous deamination of deoxyadenosine residues, which would result in T-I mis-pair [25], thus G-I mis-pair is not the major form of deoxyinosine damage that occurs in DNA. In our previous attempt to make T-I mis-pair suitable for restriction endonuclease scoring system, several restriction enzymes tested provoked extensive star activity when T-I was placed within their recognition sites [6]. In an attempt to design a T-I substrate for fine mapping of a repair patch, we found a T-I mismatch proximal to the cleavage site of restriction enzyme XcmI rendered it refractory to digestion. We first checked the integrity of T-I by sequencing and found no sign of additional sequence alteration. We then subjected this T-I substrate to repair proficient cell extracts for our repair assay and the resulting product became XcmI sensitive; the level of cleavage increased with the length of incubation throughout the 30 min time span. Due to this rather unusual property, XcmI has the potential as a scoring restriction endonuclease for evaluating the correction of T-I heterologies. We did a titration of XcmI

C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

to T-I and its canonical substrate and found that for 0.1 ␮g normal substrate, more than 98% of input substrate could be digested by 0.1 U XcmI in a reaction at 37 ◦ C for 60 min. In a similar reaction condition, less than 4% of T-I was sensitive to XcmI digestion; and even when the amount of XcmI was increased to 5 U, less than 8% background digestion of T-I was observed. Therefore, using 0.1 U of XcmI and 0.1 ␮g of substrate DNA, the XcmI could reasonably discriminate between a normal base pair and a T-I mismatch adjacent to its cleavage site. Under nitrous stress, deamination may occur in the dATP pool to form dITP, therefore deoxyinosine may also occur in DNA by mis-incorporation of dITP during replication [26]. The chemical structure of inosine is extremely similar to guanosine, which makes it base-pair preferentially with dCTP forming a C-I mispair in DNA [27]. In a much rarer event, A-I may possibly form by DNA polymerase mis-incorporation likewise forming a mismatch. Thus C-I and A-I substrates may be of biological interest to construct, to compare their repair specificity with G-I and T-I substrates. The potentially mutagenic A-I mispair was constructed using f1PMA replication form DNA as a vector and inserted into a unique PflMI recognition sequence (Table 1) to render this substrate refractory to PflMI digestion. For C-I mispairs, we tried deoxyinosine-containing oligonucleotides to complement various restriction enzyme recognition sequences forming C-I heterologies within XhoI, Bsu36I, NheI, and XcmI sites. However, it appeared that a C-I mis-pair provoked extensive star activity in the restriction enzyme reactions making these enzymes unsuitable for scoring the correction of C-I heterologies (data not shown). Since hypoxanthine incorporation forming C-I in DNA was thought to be nonmutagenic [28], we decided not to include C-I in this study. We subjected covalently-closed-circular A-I, G-I, and T-I substrates for endo V nicking assay. We found all the deoxyinosinecontaining substrates were good substrates for endo V nicking. In a near equal molar ratio of endo V and substrate condition, more than 80% of input substrates were nicked by endo V in 5 min reaction, and increasing incubation time to 50 min would drive A-I, G-I, and T-I nicking to near completion (more than 97%, data not shown).

3.2. Processing mutagenic deoxyinosine lesions in bacterial cell-free extracts We first used cell-free extracts from an E. coli strain AB1157, which have no known DNA repair deficiency, to test the newly made deoxyinosine substrates for repair. As shown in Fig. 2A and Table 3 (AB1157), A-I, G-I and T-I can be efficiently repaired and repair levels were comparable to our previous studies [6]. We then subjected these deoxyinosine substrates to endo V deficiency extracts and found that the repair levels all dropped to near background (Fig. 2B, lanes 1, 3, and 5; and Table 3, JW5547, nfi). By supplementing the purified recombinant endo V to the repair reactions, the repair levels were all significantly restored (Fig. 2B, lanes 2, 4, and 6; and Table 3, JW5547 + endo V). The correction levels of A-I and T-I substrates were comparable to our previous G-I study [6]. This result indicated that the endo V repair pathway is able to process all the mutagenic deoxyinosine lesions of A-I, G-I and T-I. In our previous study, the result using a G-I containing substrate and polA (CM5409) mutant extracts suggested that E. coli pol I was required for repair DNA synthesis [6]. Similarly as observed in this work, the repair levels of A-I, G-I and T-I in polA extracts were near background levels (Fig. 2C and Table 3, CM5409) while the repair levels of A-I, G-I, and T-I were restored to the wild type extracts levels when supplement the polA extracts with purified pol I (Fig. 2C and Table 3, CM5409 + pol I) further confirming pol I is the polymerase activity required for the repair.

903

Previous reconstitution reactions with purified proteins using pol I and its 3 -exo deficient variant also suggested the 3 -5 proofreading exonuclease of pol I was an integral part of the repair machinery for deoxyinosine excision [6]. However, there was not enough evidence to confirm this hypothesis. In this study, we employed KA796D424A (polA 3 exo-), a polymerase-proficient polA mutant specifically defective in the 3 -5 exonucleolytic proofreading activity. In the polA-D424A mutant protein, the aspartic acid residue 424 was replaced by alanine within the exonuclease active site, which is specifically defective in the 3 -5 proofreading exonuclease activity [17,29,30] while maintains its polymerase activity. As shown in Fig. 2D and Table 3, the repair levels of A-I, G-I and TI were significantly reduced in KA796D424A (polA 3 exo-) extracts compared to proofreading exonuclease-proficient KA796 (wildtype) extracts. When supplement the KA796D424A extracts with purified pol I, the results were somewhat different from the data of CM5409 + pol I (Table 3). Addition of 0.05 U pol I to KA796D424A extracts, the repair levels of A-I, G-I, and T-I were only marginally increased. As the exogenous pol I increased to 10 U, repair levels of G-I, and T-I were restored to more than half of the wild type extracts levels and A-I was restored to one third of wild type extracts levels (Table 3). It is possible that in our concentrated extracts, high level of polA-D424A mutant protein might bind to endo V nicking products and subsequently inhibited purified pol I repair reaction. Therefore, high concentration of purified pol I was required for this competitive reaction. Nevertheless, a complementation assay of KA796D424A (polA 3 exo-) and JW5547 (nfi) concentrated extracts showed the repair levels were significantly restored. This result clearly indicated the importance of 3 -5 exonuclease activity of pol I in endo V-mediated repair pathway.

3.3. Reconstitution of endo V-mediated excision repair for T-I, G-I and A-I heterologies Based on the results of our cell-free extracts assay described above, it appeared that endo V is required for substrate nicking, and pol I is required for repair synthesis and sufficient for lesion excision. Therefore, it appears that our previously suggested minimally reconstituted endo V repair reaction that includes E. coli endo V, pol I and ligase for G-I repair [6] might be complete for all the necessary and sufficient components. Here, we lowered the concentration of components in the reconstitution reaction which may better reflect the actual in vivo conditions. We found that reconstitution reactions containing 2.1 nM substrates, 1.6 nM of endo V, 20 nM pol I (0.05 U per reaction), 2 U of DNA ligase, and 0.125 mM each of four dNTPs were sufficient to repair all the deoxyinosine lesions, and more than 50% of the substrates were repaired in 30 min reactions (Fig. 3A, lanes 2, 7, and 12). For the endo V nicked circular deoxyinosine-containing DNA, it is possible for pol I to excise deoxyinosine through extensive nick translation. In order to clarify this issue, we also employed two pol I variants that lacked 5 -3 exonuclease activity in the reconstitution assay. We first tested Klenow fragment, a proofreading proficient variant and found all the substrates could be repaired with different efficiencies (Fig. 3A, lanes 3, 8, and 13). There were 10–70% decreases in repair levels compared with those reactions containing wild type pol I (Fig. 3A, lanes 2 versus 3, 7 versus 8, and 12 versus 13). We then used 3 -5 exonuclease-deficient Klenow fragment for the reactions, and the repair of all the A-I, G-I and T-I substrates decreased to background level (Fig. 3A, lanes 4, 9, and 14). Since Klenow fragment lacks a 5 -3 exonuclease activity, this observation confirmed that the repair of the deoxyinosine lesion was not the result of non-specific nick translation and clearly demonstrated that 3 -5 exonuclease domain in pol I is very important in the endo V-mediated repair pathway.

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Fig. 2. Repair of mutagenic deoxyinosine lesions in E. coli extracts. Repair reactions with E. coli extracts from different strains were determined as described in Section 2.5. DNA products were digested with AlwNI and the appropriate restriction endonuclease (Table 1) and then subjected to agarose gel electrophoresis to score for repair. The bars pointing to the 6.7-kb fragment of A-I and 7.2-kb fragment of G-I and T-I represent unrepaired substrates; and bars to 3.7 and 3.0 of A-I and 4.1 and 3.1-kb fragments of G-I and T-I indicate corrected products. (A) Lanes 1, 3, and 5 were reactions without extracts (blank). Lanes 2, 4, and 6 were repair reactions with repair proficient AB1157 extracts. (B) Lanes 1, 3, and 5 were reactions with endo V deficient JW5547 extracts. Lanes 2, 4, and 6 were JW5547 reactions supplemented with recombinant 0.05 U endo V (+ endo V). (C) Lanes 1, 3, and 5 were reactions with pol I deficient CM5409 extracts. Lanes 2, 4, and 6 were CM 5409 reactions supplemented with 0.05U pol I (+ pol I). (D) Lanes 1, 3, and 5 were reactions with proofreading exonuclease proficient KA796 polA− extracts (wt), and lanes 2, 4, and 6 were reactions with proofreading exonuclease deficient KA796 polAD424A extracts (3 exo-).

C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

905

Table 3 Repair of deoxyinosine-containing heteroduplexes with E. coli extracts. Source of E. coli extracts

Repair levels (fmol) A-I

G-I

T-I

AB1157 (wt) JW5547 (nfi) JW5547 (nfi) + 0.05 U endo V CM5409 (polA1) CM5409 (polA1) + 0.05 U pol I

15.3 1.2 14.7 3.7 14.3

± ± ± ± ±

0.49 0.23 0.26 0.61 0.92

11.4 ± 1.4 ± 12.5 ± < 0.5 13.0 ±

0.36 0.47 0.62 1.2

14.7 2.6 12.6 1.5 15.6

± ± ± ± ±

0.57 0.58 0.91 0.22 0.84

KA796 (wt) KA796D424A (polA 3 exo-) KA796D424A (polA 3 exo-) + 0.05 U pol I KA796D424A (polA 3 exo-) + 10 U pol I KA796D424A (polA 3 exo-) + JW5547 (nfi)

16.5 3.6 3.8 5.1 13.2

± ± ± ± ±

0.05 0.53 0.54 0.69 0.39

11.8 2.5 3.4 10.1 7.7

± ± ± ± ±

0.29 0.23 0.82 0.41 0.84

17.3 4.9 6.7 10.7 14.1

± ± ± ± ±

0.52 0.66 0.84 0.45 0.42

Repair efficiency was determined as described in Section 2. Deoxyinosine-containing substrate (21 fmol) was incubated with 90 ␮g of protein from indicated E. coli extracts at 37 ◦ C for 30 min. Combined cell extracts contained 45 ␮g of each extract. Each data corresponds to the average from at least three independent measurements ± 1 S.D.

A Substrate

G-I

T-I

A-I

EndoV

+

+ +

+

+ +

+

+ +

PolI

+

- -

+

- -

+

- -

Klenow

-

+ -

-

+ -

-

+ -

Klenow exo-

-

- +

-

- +

-

- +

Ligase

+

+ +

+

+ +

+

+ +

S 1

2

3

4

P

S

5

6

P 7

8

9

S

10

11 12 13 14 15

7.2 kb -

6.7 kb -

4.1 kb 3.1 kb -

3.7 kb 3.0 kb -

Repair (fmol)

11.8 6.7 <0.5

15.8 3.9 <0.5

P

18.5 13.5 <0.5

B Repair of deoxyinosine (%)

100 T-I A-I G-I

75 50 25 0 0

10 20 30 40 50 60 70 80 Time (min)

Fig. 3. Reconstitution of endo V-mediated deoxyinosine excision repair. (A) Repair reactions with indicated substrates and enzyme components were performed as described in Section 2.6. After incubating at 37 ◦ C for 30 min, reactions were terminated by heat inactivation and analyzed by restriction endonuclease digestion with AlwNI and the scoring enzymes. The (S) of lanes 1, 6, and 11 were the substrates incubated with the scoring enzymes as negative controls, and the (P) of lanes 5, 10 and 15 were homoduplex vectors treated with scoring restriction enzymes as markers for repaired products. Klenow exo- indicates the 3 -5 exonuclease-deficient Klenow fragment. The repair levels were the average from at least three independent reactions. (B) Endo V concentration was adjusted to sub-substrate level to study its turnover in the reaction. Reactions were scaled up and 10-␮l samples were removed as indicated. In each reaction, 2.1 nM A-I (triangle), G-I (square) and T-I (circle) substrates were incubated with 0.2 nM endo V and 2.0 nM pol I. Repair levels were the average of three determinations and the error bars represent 1 S.D.

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Endo V is known to remain tightly bound to nicked deoxyinosine lesions after strand cleavage [13]. Because endo V does not turn over during an in vitro incision assay, it is usually used at substrate-level concentrations to optimize the reaction. It is difficult to demonstrate turnover of endo V at this molar ratio. Although in a previous study we demonstrated that endo V would turnover in G-I reaction [6], we have yet to determine whether endo V would turnover similarly in processing A-I and T-I substrates. Therefore, we lowered endo V concentration and prolonged reaction time to confirm its turnover in the reactions. As shown in Fig. 3B, lowering the molar ratio of the endo V: deoxyinosine substrates to 1:10, still resulted in over 60–75% of deoxyinosine lesions being repaired after a 80 min reaction for all the A-I, G-I and T-I substrates which implied that endo V had to turnover at least five to six times to reach the final repair levels. 3.4. Construction of a heteroduplex in which a mismatch resides in a disrupted restriction endonuclease recognition site To understand how the 3 -5 exonuclease activity of pol I contributes to DNA repair, we designed heteroduplexes containing a mismatch at the 3 penultimate of a single-stranded DNA nick, a configuration that represents an intermediate in the repair of deoxyinosine-containing DNA through incision by E. coli endo V. Starting from phage f1PM, we prepared a set of four f1 derivatives that contain single-base differences (Fig. 1B and Table 2). This set of f1 derivatives permits construction of heteroduplexes representing all 12 possible nucleotide mis-incorporations. In each of these heteroduplexes, the heterology is located at the same position (coordinate 5856). Moreover, as shown in Table 2, each mismatch is located within overlapping restriction endonuclease recognition sites and in every case, the rest of the sequences are identical. This set of heteroduplexes allows proofreading activity on the primer to be directly determined under conditions in which the effects of the remaining sequence is conserved and hence additional proofreading effects are minimized. In order to mimic primers containing 3 penultimate mismatches, the designed DNA constructions contained a restriction endonuclease BsaI recognition sequence which can be used to incise at the second phosphodiester bond 3 to the proposed mismatch sites on complementary strand. Each DNA substrate was designated via the two relevant mismatched nucleotides, e.g. the T-G substrate (Table 2), wherein the T is the nucleotide on the template, and the G represents the mis-incorporated nucleotide at 3 penultimate site of the primer. Digestion of the heteroduplex DNA with AlwNI (Fig. 1B) and the indicator restriction endonuclease, whose recognition site was inactive because of the heterology, yielded a 6.7-kb fragment only (Fig. 4A, lane 7). In contrast digestion of the DNA in which the recognition sequence of the indicator site had been restored by proofreading yielded 3.0- and 3.7-kb fragments (Fig. 4A, lanes 1–6) In the absence of correction, all heteroduplex substrates were refractory to the digestion by the indicator restriction endonucleases with few exceptions, several heteroduplexes showed minor star activity with low level of background cleavage: A-A (5%), G-G (5%) and G-T (9%). 3.5. Correction of 3 penultimate mismatches of the primer by pol I Our previous reconstitution reactions with purified proteins using G-I substrate were performed at higher concentration of 1 mM of each dNTPs [6]. It is also well known that in the absence of a sufficient concentration of dNTPs, the 3 -5 exonuclease activity of pol I will catalyze step wise degradation from a free 3 hydroxyl end of double-stranded homoduplex DNA in vitro [22]. As a follow-up

study of previous work and to exclude the possibility of non-specific 3 degradation, we decided to use higher concentrations of dNTPs for our in vitro correction assay (1 mM, about 4–8 times higher than the optimum concentration we titrated in this study). Thus the proofreading activities were tested under conditions more favorable for DNA synthesis than DNA degradation. The proofreading activity of pol I toward 3 penultimate mismatches was performed with all 12 mismatches prepared. Using serial dilutions of pol I, suitable conditions for specificity comparisons of different mismatches were explored. As shown in Fig. 4, in 3 min reactions, each of the 3 penultimate mismatches was subjected to different proofreading levels. We found that A-A, G-A, C-A and G-G mismatches were better corrected; whereas the C-C, G-T, and T-G were poorly corrected (Fig. 4C). We did mixing substrates experiments to confirm the variation of correction efficiency within this set of heteroduplexes was not due to the presence of inhibitors in our substrate preparations. Selected substrates of C-A, C-C, C-T, T-C, T-G, and T-T that using different scoring restriction endonucleases were each incubated with A-A heteroduplex for pol I proofreading assay. These experiments yielded a pattern of correction efficiencies qualitatively similar to that obtained for the individual heteroduplexes (data not shown). 3.6. Correcting 3 penultimate mismatches of the primer is not by non-specific nick translation Although variable correction efficiencies of different mismatches at lower concentrations of pol I strongly suggested the reaction was a bona fide proofreading activity for our circular substrates, it was formally possible that removal of 3 penultimate mismatch was by non-specific nick translation activity along the circular substrate by input pol I. To further clarify this issue, we linearized the circular A-A and C-A substrates with AlwNI to avoid non-specific nick translation along the circular DNA. When these linear substrates were subject to pol I treatment, we found that the proofreading of C-A and AA mismatch in linear DNA were as efficient as with circular substrate (data not shown). Theoretically, the nicked heteroduplexes we prepared required functional 5 -3 exonuclease for nick translation to restore the complete restriction endonuclease sites for scoring. To evaluate proofreading without 5 -3 exonuclease intervention, we devised a gapped heteroduplex which containing a 24-nt single-stranded region 3 to the primer with a penultimate G-G mismatch, as illustrated in Fig. 5A. As shown in Fig. 5B (white bar versus gray bar), pol I demonstrated similar repair levels for gapped and nicked G-G heteroduplex. We then tested the proofreading activity of Klenow fragment with the gapped G-G substrate. Our results showed Klenow was also capable of correcting the G-G mismatch at 3 penultimate position of the primer (Fig. 5B, black bars). This further assured the correction we observed was not due to the consequence of nonspecific nick translation. However, the proofreading efficiencies of Klenow fragment were lower than pol I (Fig. 5B, black bars versus white bars). In a reconstitution assay for deoxyinosine repair, we also observed that Klenow fragment was less effective than complete pol I (Fig. 3). A previous report indicated that removal of a protein domain of a polymerase may affect its fidelity [31]; it is also possible that a complete pol I structure is required for its full capacity in the repair process. However, it should be noted that in our correction assay, while the concentrations of pol I and Klenow fragment were adjusted based on units of the polymerase activity indicated by the manufacturer, the correlation of units of the 3 -5 exonuclease activity and the units of the polymerase activity of these two

C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

A

1

2

3

4

5

6

907

7

8 6.7 kb

A-A

G-G

5

01 0.

1

00

0.

00

05

0.

T-C

25 20 15 10 5 0 25 20 15 10 5 0

T-G

Pol I (unit)

5

01 0.

00

0.

00 1

0.

05 00

0.

0.

00 00 0.

01

5

T-T

01 0.

0.

00

5

1 00

05

0.

00

01 00

0.

0.

00

00

5

C-T

25 20 15 10 5 0

00

Correction (fmol)

C-C

0.

Correction (fmol)

0.

Pol I (unit)

C-A

25 20 15 10 5 0

00

01

00 00 0.

00

5

G-T

Pol I (unit)

25 20 15 10 5 0 25 20 15 10 5 0

0

5 00

0. Correction (fmol)

01

00 0.

0.

5

1 00

00

05

01 00

0.

0.

0.

00

00

5

A-G

G-A

25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0

0.

25 20 15 10 5 0

00

01 00

00 0.

A-C

0.

1 00

5

0.

00

01

0.

A-A

25 20 15 10 5 0 25 20 15 10 5 0

0.

Correction (fmol)

B

0.

Pol I (unit)

05

3.7 kb 3.0 kb

Pol I (unit)

C *

* * *

Correction (fmol)

*

* * *

** ** ** ** **

** ** ** * **

** ** ** ** **

** ** ** ** ** * * **

** ** ** ** ** * * *

A-A G-A C-A G-G T-C T-T A-C C-T A-G T-G C-C G-T

Mismatch Fig. 4. Concentration dependency of pol I proofreading toward all twelve 3 penultimate mismatches. Proofreading reactions with indicated concentrations of pol I were determined as described in Section 2.7. Each reaction contained 23 fmol substrate and incubation time was 3 min. (A) A gel example from A-A heteroduplex, DNA products were digested with AlwNI and PflM1 and then subjected to agarose gel electrophoresis to score the proofreading. The bar pointing to the 6.7-kb fragment represent unrepaired substrates; size marks (M) of 3.7- and 3.0-kb fragments indicate repaired products were generated by treatment of f1PMA with AlwNI and PflM1I. (B) Quantitative analysis of pol I proofreading of all twelve possible mismatches, repair levels were the average of three determinations and the error bars represent 1 S.D. (C) Statistic analysis of pol I proofreading specificity, results shown were the Student’s t-tests of the measurements from (B) entries of 0.0005 U data. *p < 0.05 versus the better repaired substrate; **p < 0.01 versus the better repaired substrate.

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C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

A V C

Cpo I 5'-............CTAGGGTCTCCGGACCGTAA...... 3'-... 24-nt gap GGATT...... 3' 5' 5' 3' C

V

B pol I + gapped G-G Correction (fmol)

25

pol I + nicked G-G

Table 4 Correction efficiencies of 3 penultimate mismatches and deoxyinosine lesions by pol I. Heteroduplex

Rate of correction v0 (fmol s−1 )

A-A A-C A-G C-A C-C C-T G-A G-G G-T T-C T-G T-T

0.063 0.065 0.059 0.072 0.019 0.029 0.054 0.079 0.018 0.049 0.017 0.037

A-I T-I G-I

0.022 0.046 0.013

Results shown are based on initial rate measurements from the experiments presented in Fig. 6 and represent the average of at least three determinations.

KF + gapped G-G 20 *

*

15

The hierarchy for the initial rates calculated were as following:

*

*

G-G > C-A > A-C > A-A > A-G > G-A > T-C > T-T > C-T > C-C > G-T > T-G

10

Generally, the purine–purine mismatches were better corrected than pyrimidine–pyrimidine mismatches. For the purine–pyrimidine mismatches, C-A and A-C were corrected as efficiently as the purine–purine mismatches, while G-T and T-G mismatches were least corrected of all.

5 0

0.

0 00

05

0 0.

00

1 0 0.

00

5 0.

00

1

5

00

0.

01

0.

Pol I (unit) Fig. 5. Proofreading of pol I on nicked-circular and gapped-circular substrates. Proofreading reactions were determined as described in Section 2.7. (A) Illustration of gapped G-G heteroduplex. (B) In each reaction, 23 fmol G-G containing substrate was incubated with indicated pol I at 37 ◦ C for 3 min. Reactions were stopped by heat inactivation and proofreading products were digested with AlwNI and CpoI to score the repair. White bars are the reactions of gapped G-G substrate, gray bars are the reactions of nicked G-G substrate, and black bars are the reactions of Klenow fragment with gapped G-G substrate. Repair levels were the average of three determinations and the error bars represent 1 S.D. *p < 0.05 versus gapped G-G proofread by pol I (Student’s t-test).

enzymes have yet to be determined. Therefore, whether a complete pol I structure is important for its full capacity for proofreading and repair requires further study for confirmation. 3.7. Specificity analysis of proofreading of a 3 penultimate mismatch kinetic assay Based on enzyme titration data from Fig. 4, correction efficiency varied when pol I was altered from 0.00005 to 0.001 U with 23 fmol heteroduplexes. Thus, a kinetic analysis with 0.0005 U of pol I was performed to compare substrate specificity where the enzyme versus substrate ratio was about 1:10. As shown in Fig. 6A–D and summarized in Table 4, members of this set were corrected by pol I with different efficiencies. Heteroduplexes of G-G and C-A were efficiently corrected while G-T and C-C substrates were corrected at much reduced rates. In fact, there was more than a 4-fold difference in the rate of repair between the best-corrected heteroduplex and the least corrected substrate.

3.8. A deoxyinosine at the 3 penultimate site of the primer is corrected as efficiently as a mismatch To confirm our hypothesis that the deoxyinosine at the endo V nicking site showed great similarity to a mis-incorporated nucleotide which provoked pol I excising it as part of its proofreading function, we compared proofreading activity of mismatches versus deoxyinosine substrates. Due to the constraints of restriction enzyme selection for deoxyinosine repair scoring [6], we placed A-I, G-I and T-I mismatches in different sequence contexts. Although local sequence environment could have affected the proofreading efficiency, we observed corrections of A-I, G-I and T-I with efficiencies comparable to our mismatches. As shown in Fig. 6E and Table 4, the correction rate of G-I was about half to one third that of G-A or G-G proofreading. Proofreading of T-I was more efficient (Fig. 6E and Table 4), with the rate more than twice that of the T-G correction. We also performed the deoxyinosine corrections with 125 ␮M each of the four dNTPs. As shown in Fig. 6E and F, different concentrations of dNTPs did not affect the general order of substrate specificity of deoxyinosine corrections. 4. Discussion The most significant finding from this study is that 3 -5 exonuclease activity associated with pol I is important in the processing all three mutagenic deoxyinosine lesions in cell-free extracts. There has been speculation regarding which component(s) are involved in the processing of endo V nicking product before repair DNA synthesis [5,13]. In this study, we used 3 -5 exonuclease deficient polA mutant strain providing solid evidence to show 3 -5 proofreading exonuclease of pol I is a major component in the repair pathway. In this study we added other two mutagenic deoxyinosine containing substrates T-I and A-I to our assay platform for the endo V

C.-C. Lee et al. / DNA Repair 12 (2013) 899–911

(A)

(B)

25

20

A-A A-G

15

A-C

10 5

Correction (fmol)

Correction (fmol)

25

20 C-A

15 10

C-T C-C

5 0

0 0

2

4

0

6

Time (min)

2

6

(D)

20 G-A G-G

15 10

G-T

5

Correction (fmol)

25 20

T-T T-C

15 10 5

T-G

0

0 0

2

4

0

6

Time (min)

2

4

6

Time (min)

(E)

(F) 25

20 15

T-I

10 A-I G-I

5

Correction (fmol)

25 Correction (fmol)

4

Time (min)

(C)

25 Correction (fmol)

909

20

C-A

15

T-I

10

A-I G-I

5 0

0 0

2

4

6

Time (min)

0

2

4

6

Time (min)

Fig. 6. Kinetic analysis of pol I proofreading on 3 penultimate mismatches. Proofreading reactions were determined as described in Section 2.7. DNA substrates utilized are those shown in Tables 1 and 2 of different 3 penultimate mismatches. The pol I used was 0.0005 U per 10 ␮l reaction. Samples were removed at indicated times and reactions were stopped by heat inactivation. DNA samples were scored with restriction endonucleases as shown in Tables 1 and 2. Repair levels were the average of three determinations and the error bars represent 1 S.D. (A) Reactions of A-A, A-C, and A-G. (B) Reactions of C-A, C-C, and C-T. (C) Reactions of G-A, G-G, and G-T. (D) Reactions of T-C, T-G, and T-T. (E) Reactions of endo V nicked A-I, G-I, and T-I with 1 mM each of the four dNTPs. (F) Reactions of C-A, endo V nicked A-I, G-I, and T-I with 125 ␮M each of the four dNTPs.

repair pathway. Under similar reaction conditions, we found both T-I and A-I are better substrates than G-I both in cell-free extracts (Fig. 2 and Table 3) and our reconstitution assay (Fig. 3). And in the correction assay of 3 penultimate errors by pol I, T-I also showed the best correction result among A-I, G-I and T-I substrates (Fig. 6 and Table 4). Since T-I is the most biological relevant deoxyinosine containing substrate [3], combining the property of higher sensitivity for correction renders it the substrate of choice for the endo V-mediated repair assay. In order to compare the catalytic specificity toward substrates similar to endo V nicking intermediates, we developed a set of heteroduplexes with 3 penultimate mismatches embedded in primers

of similar sequence environment. For the convenience of repair scoring we placed all 12 possible mismatches within overlapping restriction endonuclease recognition sites. We found that pol I was capable of correcting all 12 mismatches. The efficiencies of proofreading by pol I of the 3 penultimate mismatches covered a more than 4-fold range (Table 4), where purine–purine mismatches generally were better substrates than pyrimidine–pyrimidine or purine–pyrimidine mismatches suggesting that two mismatched bulky purines may generate more distortion in double helix resulting in a poorer fit in the polymerization site. Thus purine–purine mispairs should have a higher tendency to be transferred to 3 -5 exonuclease site.

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Our results share some similarity to a previous report of 3 end proofreading by Klenow fragment [32]. These authors showed mismatches were excised within a narrow range of rates with no more than 3-fold difference. The general order of editing also showed excision of purines was more efficient than pyrimidines. The authors further suggested that purines might interact with the exo site residues more favorably because of their increased stacking ability, or by their common N7 groups [32]. Another possible selection for specific mismatches could result from the formation of novel hydrogen bonds and spontaneously pairing in rare tautomeric forms. Two possible transition mispairs, A-C and G-T, involve the enol form of guanine or thymine and the imino form of adenine or cytosine, respectively. Both mispairs fit well within the dimensions of the DNA double helix to preserve the geometry of a correct Watson–Crick base pair [33,34]. It is possible such a purine–pyrimidine mismatch in the 3 penultimate position of the primer is stable enough to escape proofreading. Consistent with this notion both T-G and G-T heteroduplexes were poor substrates in our proofreading assay. In contrast this result differs from previous mismatch repair studies; T-G and G-T were generally very good substrate for MutS in E. coli [35]. It is possible that such a deficit in DNA replication and proofreading for the T-G and G-T heteroduplexes was a force behind the evolution of MutS-mediated mismatch repair to ensure genome integrity. C-A was an exception of purine–pyrimidine mismatches and demonstrated poor correction by pol I. As shown in Fig. 6 and Table 4, C-A at 3 penultimate site was one of the best substrates for correction by pol I. A detailed analysis of the local sequence of C-A substrate showed that mis-incorporated adenosine could adopt an extra-helical conformation by terminal guanine pairing with cytosine (Table 2). It has been shown that the presence of an extrahelical base at the first position from the primer 3 terminus increased the level of partitioning of the DNA substrates into the 3 exonuclease site by 3- to 7-fold, relative to the perfectly base-paired primertemplate [21]. Furthermore, purines at extrahelical positions were better substrates than pyrimidines to promote partitioning of DNA into the 3 -5 exonuclease site [21]. This may explain why our C-A heteroduplex provoked such high level of proofreading by pol I. This work broadens our knowledge of pol I proofreading capability. Previously, it was assumed that a correct nucleotide was added to a mis-incorporated nucleotide during replication, and the processive DNA polymerase would continue new DNA synthesis, leaving a mismatch in the newly polymerized sequence. Here we provide new evidence that DNA polymerase gets a ‘second chance’ to correct the penultimate mismatch of the primer through proofreading. We also subjected endo V nicked A-I, G-I and T-I substrates to our pol I proofreading assay which yielded efficiencies comparable to mismatches (Fig. 6). Our results showed that T-I is a much better substrate than A-I and G-I, being corrected by pol I two times more efficiently as measured by initial rate kinetics (Table 4). The high efficiency of T-I proofreading by pol I was consistent with the results from both cell extracts and reconstitution assays. However, the proofreading of A-I was relatively poor which deviated from the results of complete repair assays. It was speculated that endo V remains bound to nicked deoxyinosine lesion after strand cleavage and may contribute to excision promotion or regulation [13]. Further study is required to understand whether there are protein–protein interactions between endo V and pol I in promoting the deoxyinosine repair process. Here we also provided evidence for our earlier hypothesis that the 3 -5 exonuclease activity of pol I was the major exonuclease required for endo V-mediated alternative excision repair of deoxyinosine [6]. It is well known that E. coli pol I can be utilized as a 3 -5 exonuclease in vitro. However, pol I will remove no net nucleotides from the 3 end of a strand break in the presence

of the four dNTPs in perfect matched primer-template sequences. Here we find that a mismatch at a 3 penultimate site of the primer may also destabilize the 3 end, thereby making it a substrate for the single-strand specific 3 -5 exonuclease activity of pol I and blocking its primer activity until the wrong nucleotides are removed. Our data also show that correction rates of pol I to deoxyinosine lesions and mismatches at 3 penultimate site are comparable (Table 4). These experiments strongly suggest that removing deoxyinosine lesions by 3 -5 exonuclease of pol I is a part of its native capability for proofreading. Our results do not rule out the possible involvement of other exonuclease/endonuclease in removing deoxyinosine, since there was a residual repair activity of 3 -5 exonuclease deficient polA extracts (Table 3, KA796D424A (polA 3 exo-) entry). This low repair activity may be coming from unidentified exonuclease/endonuclease associated with mutant pol I or other redundant but minor repair mechanism(s). Based on our results, we propose a multistep working model of endo V-mediated repair. Here we suggest that repair of deoxyinosine is initiated by endo V generating a strand break at the second phosphodiester bond 3 to the lesion [13]. The subsequent removal of the damaged nucleotide is performed by the proofreading 3 -5 exonuclease activity of pol I, which makes limited excision to generate a small gap of at least 3-nt [5]. Finally, the gap is filled and sealed by pol I and DNA ligase respectively. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Science Council, Taipei, Taiwan, ROC [NSC 100-2320-B-002-103]. References [1] T. Nguyen, D. Brunson, C.L. Crespi, B.W. Penman, J.S. Wishnok, S.R. Tannenbaum, DNA damage and mutation in human cells exposed to nitric oxide in vitro, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3030–3034. [2] T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715. [3] M. Hill-Perkins, M.D. Jones, P. Karran, Site-specific mutagenesis in vivo by single methylated or deaminated purine bases, Mutat. Res. 162 (1986) 153–163. [4] G. Guo, B. Weiss, V. Endonuclease, (nfi) mutant of Escherichia coli K-12, J. Bacteriol. 180 (1998) 46–51. [5] B. Weiss, Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation, DNA Repair (Amst) 7 (2008) 205–212. [6] C.C. Lee, Y.C. Yang, S.D. Goodman, Y.H. Yu, S.B. Lin, J.T. Kao, K.-S. Tsai, W.H. Fang, Endonuclease V-mediated deoxyinosine excision repair in vitro, DNA Repair (Amst) 9 (2010) 1073–1079. [7] W. Cao, Endonuclease V: an unusual enzyme for repair of DNA deamination, Cell. Mol. Life Sci. 70 (2013) 3145–3156. [8] S. Riazuddin, T. Lindahl, Properties of 3-methyladenine-DNA glycosylase from Escherichia coli, Biochemistry 17 (1978) 2110–2118. [9] P.J. O’Brien, T. Ellenberger, The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site, J. Biol. Chem. 279 (2004) 26876–26884. [10] K.A. Schouten, B. Weiss, Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid, Mutat. Res. 435 (1999) 245–254. [11] F.T. Gates, S. Linn, Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, acid, or X-rays, J. Biol. Chem. 252 (1977) 2802–2807. [12] M. Yao, Z. Hatahet, R.J. Melamede, Y.W. Kow, Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3 endonuclease, from Escherichia coli, J. Biol. Chem. 269 (1994) 16260–16268. [13] M. Yao, Y.W. Kow, Interaction of deoxyinosine 3 -endonuclease from Escherichia coli with DNA containing deoxyinosine, J. Biol. Chem. 270 (1995) 28609–28616. [14] R. Mi, M. Alford-Zappala, Y.W. Kow, R.P. Cunningham, W. Cao, Human endonuclease V as a repair enzyme for DNA deamination, Mutat. Res. 735 (2012) 12–18. [15] C.M. Joyce, N.D. Grindley, Method for determining whether a gene of Escherichia coli is essential: application to the polA gene, J. Bacteriol. 158 (1984) 636–643.

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