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Postreplication repair of DNA in mammalian cells
Life Sciences Vol . 15, pp . Printed is the II .S .A .
Pergamon Press
2005-2016
MINIREVISiO P081RSPLICATION R1~AIR OF DNA IN 1fA18fALIAN CBLLB Alen R .
nnn
T
Y .R .C . Cell Mutation Unit, University of 8useex, Falmer, Brighton HN1 94G, Bagland
Ys®alien calls possess vecheniema for rmoving or circumventing various lesions is their DNA, and this has been the eub~ect of a review in this aeries by 8trause (1) .
Many lesions remain in the DNA in damaged cells, without pre Poetreplication repair of DNA may be
venting replication irov taking place .
defined is its broadest sense ae the manner is which the DNA replication machinery copes with lesions in the parental DNA strands .
Ii damaged bases
are copied incorrectly, this may lead to cell death, mutation and possibly carcinogeneeie .
The process ie therefore of great importance .
It will be die-
cussed principally with regard to davege ceased by ultraviolet (W) irradiation . Strictly speaking it ie not a repair process, since the lounge ae such ie not removed .
It ie closely associated with DNA replication .
Bacteria can remove W-induced cyclobutane pyrimidine divers cram DNA (a) by photoreactivation, a light-induced splitting of pyrimidine diniere in situ , which has not been detected in vivo is placental eiammale (reviewed in Ref . (2)), ar (b) by excision repair, which oleo occurs in human cells (3) but only to a very limited extent in rodent cells .
Despite this poor ability of marry mamvslian
cells to remove pyrimidine divers, they can in general survive W flumcee which produce 105 -108 such lesions per cell .
Poetreplication Repair in Bacteria In strains of Becherichia coli unable to excise pyrividine diners Rupp and 2005
2006
Postreplication Repair of DNA in Mammalian Cells
Vol. 15, No . 12
Howard-Flanders ßcund that newly-synthesized DNA in W-irradiated cells was initially oß lower molecular weight than is unirradiated cells (4) .
This
molecular weight oß the DNA gradually increased on subsequent incubation, eventually reaching the same size as that of DNA from unirradiated controls . They postulated that the DNA "replicaee" was temporarily delayed at each dimes (to account ßor the W-induced inhibition oß DNA synthesis) and then continued beyond the dieter, leaving a gap .
This gap was subsequently sealed .
In later
work, they showed that Dd~W replicated in W-irradiated cells did indeed contain single-stranded regions, about 1000 nucleotides in length (5) .
ibccellent
indirect evidence has been obtained that these single-stranded regions are the result oß gaps opposite the dieters (B-S) .
The gap-Billing is achieved by a
process involving mates-strand recombinational exchanges oß large amounts oß DNA (9, 10) and recent evidence indicates that the dieters (ae measured by sites susceptible to the W-specißic T4 endonucleaee-V) become equally distributed between parental and daughter strands (11) . In animal cells, a similar process seems to occur, marked difßer~cde in details.
although there are some
The writing oß this review comes at a time when
some oß the interpretations oß the available data ere being questioned, so careßu1 attempts will be made to distinguish ßacta and experimental results, ßrom interpretation and speculation .
Interpretations presented will naturally
reflect my personal bias, and should therefore be treated with appropriate caut ion.
Ilechaniema oß DNA Replication in Damaged Cells D3QA in mammalian chromoe~ea is synthesized by many independent replicating forks.
Replication occurs b1d1ractione1ly (12,13) ßrom multiple origins at a
rate oß 0" 5-2 " 5 N/min per replication Bark . origins vary ßrom 16 to 200 microns .
Setimatee oß the distance between
After replication adjacent replication
segments (replicona) are joined together to produce very long DNA strands . As with bacteria, it has been shown in many mammalian cell lines, that W
Vol. 15, No . 12
Poatreplicatioa Repair of DNA is Mammalian Cells
and other insults decrease the rate of
DTiA
replicatirn .
This is presumably
a consequence of some or all of the lesions in the changed DNA
replication machinery .
200 7
Dd~iA
delaying the
The fraction of pyrimidine diners in
DiNA
repli-
cated after W-irradiation of Chinese hamster cells ie the same as that in unreplicated
DNA,
indicating that the dLere only delay
DNA
replication tem-
porarily and do not completely block it (14,16) . Various hypothetical schwas by which lesions in growth of daughter-strands are shown in Fig.l .
DNA
could affect the
Hyntheeis may continue r oued
the lesion (Fig . ld) possibly after a delay, it may be halted completely (Fig . 1a),
it may be delayed and then continue beyond the lesion leaving a
discontinuity opposite the lesion (Fig .
Ib) .
Thin say be sealed some time
later ae is Fig . Ib or very shortly after its formation (Fig . lc ) . FIO. 1
O
b
o
.
. . "n
..
0
C
d
O
O
O
0 o u
00
0
.
o0
0 0
o
a
"o
O
Ct
.
Fig. 1 . Yodels for replication on damaged templates ; a - lesions completely block replication ; b - gape left opposite lesions and filled in ease time later; c - as b, but gape filled in rRpidly; d - continuous synthesis peat lesions . 0, origins of replication ; ~, lesions ; ~, ü lled in gap ; -, parental DNA ; ~, newly-synthesized DNA .
Bize of
DNA
Made is W-Irradiated Cells
Yo st of the work on poetreplication repair in mammalien celle hoe involved meas~emeata of the mol . wt, of DNA from lysed celle, using the technique of centrifugation in alkaline sucrose gradients (18) .
The study of
D1~iA
of mol .
2008
Postreplication Repair of DNA in Mammalian Cells
vol. 15, No . 12
wt . greater than 5 x 108 ie fraught with dißßiailtiee caused by incomplete denaturatioa, and aggregation and entanglement effects, whidi are little understood .
In order to overcome these problems, steps have usually been taken to
fragment the DNA to mol . wts . below 5 x 10 8 immediately before centrifugation . This has been achieved by prolonged lyeie treatments in alkali, or by low doses oß X- or Y-irradiation . Sxperimente by many workers, using alkaline sucrose gradients,
have shown
that is several Chinese hamster and mouse cell lines, as in bacteria, newlysynthesized DNA (labelled by a pulse oß 3H-thymidine) in W-irradiated cells is smaller than that in unirradiated cells (17-22) .
8lmilar observations have
been made with normal human cells, and cells derived from patients carrying the inherited akin disease xeroderma pigmentoeum (XP) (23), a disorder characterized by a deficiency in the ability oß the cells to excise W-induced pyrimidine diniere from their DNA (24 ). ßluences oß 7-100
Jm 2,
The above experim~te were carried auf using W
and were interpreted on the bacterial model, ae evidence
far discontinuities in the daughter-strands opposite pyrimidine diniere (Fig . lb). In order to eliminate the possibility that the results are merely due to the slower rate oß synthesis in W-irradiated cells several oß the above walkers pulse-labelled irradiated cells ßas longer times than the unirradiated controls, so that the same amount oß radioactivity was incorporated in unirradiated and irradiated cells. Some evidence hoe been obtained for single-stranded regions in replicated DNA from W-irradiated sauee cells (25), similar to those.ßound in S.ooli (6). The size oß short pieces oß newly-synthesized i~iA is approximately equal to the average distance between dimes in the parental strands, as calculated from the fraction oß thymine in pyrimidine diniere (21,23,25) .
Thin ie caaeietent with
the idea that the gaps in the new DNA are opposite the diniere.
It should, how-
ever, be pointed out that both the above parauetere are eub~ect to large experimental errors and that this rather crude comparison ie the only evidence available to indicate that the gape are in fact opposite diniere in placental mammals .
Postreplication Repair of DNA in Mammalian Cells
Vol . 15, No . 12
200 9
Recently, Buhl, Setlow and Regan using a marsupial cell line, compared the size of newly-synthesized DNA in cells which had been W-irradiated to that from cells which were W-irradiated followed by photoreactivation to remove some of the diniere (28) .
In the latter case the DNA was larger than in the former sug~
Besting again that the gape wpa a opposite the dimes e .
Although photoreacti-
vesting enzyme has recently been detected in human leucocytee (27), photoreactivat ion in vivo has never been detected in placental mammals .
Hence this type
of experiment can only be carried out with marsupials and lower orders . In cotrtraet to the results discussed eo far, Chiu and Rauth found that in W-irradiated souse L cells, newly-synthesized DNA was the same size se that in unirradiated cells (28), provided that care was taken to eliminate labelling artefacts (29), by labelling equivalent lengths of DNA in irradiated and unirradiated cells .
DS~A made in cells irradiated with high W iluencea
(i 20 Jm2) was smaller than that in unirradiated cella, but the size of the pieces synthesized was cvneiderably greater than the inter-dimec~distance . These data suggested that replication continued round the dieter (Fig . id) but could also be explained if gape were left sad sealed vea~y rapidly (Fig . 1c ) . In later work Rauth et al ., compered the mol . wts . of DNA synthesized in Wirradiated L cells, CHO and HeLa cells (30) . the cell lines .
They found differences between
These could be explained by differing raYres of gap-filling in
these cell lines, or by the presence of both a discontinuous synthesis (with gape) and a continuous synthesis peat dieters . At the opposite extreue Edenberg has recently suggested that the low mol . wt . DANA seen in alkaline sucrose gradients does not represent DNA strands coatsining small gape, but that synthesis is halted far a long period at the dimmers rather than continuing beyond after a short delay (31) .
In my opinim ,
presently available data do not substantiate this suggestion (32) and far the rest of this review it will be aesuved that discontinuities are in fact left opposite dieters, end then sealed (as in Fig . Ib or Ic) although this remains to be rigorously proven .
201 0
Poetreplication Repair of DNA in Mammalian Cells
Vol . 15, No . 12
Filling in of Rape The low molecular weight DNA in UV-irradiated cells is detected by alkaline sucrose sedimentation of DNA from cells labelled with a short pulse of 3H-thymidine .
Ii this pulse ie followed by a chase in unlabelled thymidine,
it has been sound in all the cell Bass examined that the low mol . wt . DNA increases
3a size, eventually attaining a mol . wt . similar to that from
uairradiated cells (17-19,21-23) .
This increase in mol . wt . i e the result of
two processes : (a)
those associated with normal DNA replication and therefore seen eleo in
unirradiated cells (chain elongation within replicating units and joining of adjacent replicating units,
(b)
filling-in of daughter-strand gape in damaged celle .
Gap-filling is
inhibited by the DNA synthesis inhibitors hydro~urea (21,23,33), cytosine arabinoeide and excess thymidine (33), and by
actinomycin D (33), but not by
cycloheximide, as inhibitor of protein synthesis (33,34) .
In rodent cells it
ie possible to inhibit gap-filling without greatly sirecting DNA replication, with caffeine (17, 20,22,35,25,38), or its analogue
theophylline (37) .
Althougà caffeine does affect normal DNA synthesis to a small extent (38) its effect on W-irradiated cells is far more pronounced .
In the latter case,
in the presence of caffeine, low mol . wt . DNA accumulates during e prolonged labelling period (25,38), wher~a in its absence it slowly increases in size due to filling~in of the gape .
In normal human cell linen caffeine has
hardly any inhibitory effect on gap-filling (22,39-41), although under some conditions a small inhibition may be observed (40,42) .
In XP cells, however,
it has a vea~y marked effect (see below) .
These effects of caüeine correlate
very well with effects on cell survival .
Caffeine sensitizes many rodent
cells to W-irradiation (43) but only if present during the DNA synthesis period (44), and it does not sensitize human (HeIa) cells (46) .
Vol . 15, No . 12
Postreplication Repair of DNA in Mammalian Celle
201 1
Mechanism of gap-filling Ia S .coli Rupp et al ., (9) postulated that the daughter-strand gaps were filled by a process in which the genetic information lost by the formation of a gap opposite a dieter was retrieved by a sister-attend recombinational exchange . The piece of parental DNA from the sister duplex, which contained the eiesing sequence of nucleotides was inserted in the gap opposite the dieter .
The
resulting gap is the parental strand of the sister duplex, could then be filled in by repair replication, since it was opposite an intact template .
Hy tech-
niques involving density-labelling and equilibrium centrüugatioa in alkaline CeCl (9) or bromodeoxyuridine photolysie (10) evidence has been obtained for
end-to-mod association of par~tal and daughter-strand DI~iA after W irradiation indicating that exchanges of large lengths of DNA (about 6 x 108 daltons) between parental and daughter-strand had occurred . Attempts to detect exchanges in mammaliea cells have been made using photoZyais of bromodeoxyuridine-substituted DNA (21), alkaline CsCl c~trifugatioa (25,48), and detection of pyrimidine dieters in daughter-strand Dl~iA (47, 48) .
These techniques have all given positive indication of exchanges in
B .coli (9-i!), but Gave failed to detect such exchanges in mammalian cells .
Fhrthermore the daughter-strand gape seem to be sealed by de aovo synthesis with a stretch of DNA approximately 1000 nucleotides in length (21,49,48) . There is therefore at present no evidence to support the bacterial model of gap-filling by recombinational exchanges .
If such exchanges exist, they have
not beg detectable by the techniques available, and must be leas than about 100 nucleotides in length .
Certainly ~oae exchanges such as occur in S .coli
(9,10) can be ruled out .
Recovery_ of Ability to Synthesize High ![ol . i"t .- 7$iA at Ixte Times attar Irradiation All the walk reviewed so far has dealt with stretches of DNA synthesized within an hour or two after irradiation .
Hiace rodent cells have only a very low
Poatreplication Repair of DNA is Maa~alian Cells
2012
Vol . 15, No . 12
level of excision of pyrieidine dimere (50,37,36,14), it would be anticipated that all DNA synthesized within the Piret generation after W-irradiation would initially curtain gape, since the template still contained dimes . Surprisingly, however, it was shown in rodent cells that the DNA synthesized in a short pulse-label given 8 - 8 hours niter irradiation was the same size as that in unirradiated cells, i .e ., no gape could be detected (19,37) .
This
recovery of the ability to synthesize high mol . wt . DNA did not occur in a W sensitive Chinese hamster cell line, B14 (19) .
It occurred in both normal
human cells and in the excision-deficient XP cell lines at the same rate (81) . F1u~thermore, it took place even if DNA synthesis was prevented by hydroxyurea between the ties of irradiation end pulse-labelling (81) . ie a mystery .
This phenomenon
1~Scplenatione which have been put forward include :
(1)
rapid gap-Pilling at late times after irradiation (37),
(2)
the possibility that the gaps are opposite soee lesion other than pyrimidine dimere, and this lesion is excised is the First Pew hours after irradiation in all the cell lines studied (51),
(3)
a coniarmational change of the dimere such that they no longer lead to the production oP daughter-strand gaps (81) .
None of these explanations satisfactorily account Por all the available data, sad the phenomenon remains a mystery though its existence ie certainly beyond doubt .
Gap-filling in Xeroderma Pig tosum Çelle It has recently been shown that 7CP cells can be divided into at lee at five or six complementation groups on the basis oP cell Fusion experiments (82,53) . In at least three of these, gape were left in daughter-strand DNA after Wirradiation and these gape were subsequently sealed (23) .
In as XP line from
the "classical XP" complementation group both the gap-Pilling and the recovery of the ability to synthesize high mol . wt . DNA at late times after irradiation were inhibited by caffeine (39) .
Normal cells were unaffected .
Other XP
Vol. 15, No . 12
Postreplication Repair of DNA is ilaamalian Cells
2013
cosplementatica groups were not examined for caffeine sensitivity. One group o! patients have been clinically diagnosed ae having 7[P, but unlike moat of the other cases eaa
In fibro-
blast cells from three of these cases the ooavereion of low mol . wt . DNA to high ool . wt . after W-irradiation was considerably slower than in normal fibroblaeta (40) .
Furthermore this coavea~eian was drastically inhibited by
caffeine, which had very little effect on normal cells .
A "classical 7[P"
line and a line with the de-8aactis~accione syndrome showed intermediate behaviour both with regard to rate of gap-filling and inhibition by caffeine (40) .
Thus the 7[P lines normal in excision-repair have same deficiency in
postrepücation-repair, although the nat~ae of this deficiency is at present not clear .
From these ündings we have the interesting and rather surprising
observation that a deficiency in postreplication repair and various defects in excision-repair at the cellular level seem to give rise to similar clinical symptoms in man .
It seems that pyrimidine
dimere induced is human skin by
sunlight (66) are highly likely to lead to skin cancer either if the dimere are not excised (as in most XP cases) or if they are replicated in a defective manner (as in the varisate discussed above) . Poetreplicatian Repair in Cells Damaged with Ch®kale The way in which the Dd1A replication machineay copes with lesions induced by chemicals such as alkylating agents end other mutegens, has not as yet been studied in ae much detail as with W-irradiation .
Methyl methane eulphonate
(1018) ie an alkylating agent which produces a variety of lesions in D~ (68) . DNA synthesized in the first few hours after treatment with 1W8 ie abnormally short (57,68) and ae with W these abort chAine are subsequently elongated. Using
HND-cellulose chromatography to detect single-strand regions of DNA,
8cudiero and 8traues found a higher proportion of such regions in 1018-treated
Postreplication Repair of DNA in Mammalian Cells
201 4
cells than in untr~ted controls (59) . lesions blocks DNA synthesis .
Vol . 15, No . 12
They suggest that one of the induced
After removal of the lesion, replication can
continue . Based on detailed kinetic atudiea of the effects of N-methyl N-nitrosourea and sulphur mustard on DNA synthesis, Roberta and colleagues inferred that lesions i~uced by these ag~ta somehow interfere with replication, but they can be overcome poatreplicatively (60) .
Ae with W-irradiation caffeine sensi-
tized Chinese hamster cells but not HeIa cells to alkylating agents and similarly affected DNA synthesis and cell progression in the rodent but not the human line .
Preliminary molecular atudiea am the size of DNA synthesized in
Chinese hamster cells treated with N-methyl N-nitrosourea conürmed that the induced lesions acmehow interfered with the normal synthesis of high mol, wt . DNA and caffeine had a further inhibitory effect (81) .
Similarly Yan den
Berg found that treatment of HeLa cells with the carcinogen methylazoxy methanol acetate interfered with chain elongation (82) .
Finally Troeko et al .
found that in Chinese hamster cells treated with N-acetoxyaminoiluorene,se with W , caffeine inhibited the eventual synthesis of him mol . wt . DNA (83) . Thus in cells treated with alkylating agents, ae with W , there ae®s to be a caffeine-sensitive S-phase specific poatreplicative repair process .
The
mechanism seems to have acme eimiliaritiea to the dimer/gap mechani® for poatreplication repair in W-irradiated cells, but may düfer significantly in details .
Only preliminary data is available at present .
Relationship of Poetreplication Repair to Mutation, Chromosome Aberratim e and 0arçinogenesie In cells from patients with "classical XP" or the de-8anctie~accione eyndroue pyrimidine dimes are not excised, and more of these lesions moat be "repaired" poatreplicatively than in normal cells .
Since patients with XP inevitably
develop akin carcinomas, it has been speculatively suggested that postreplication repair is an error-prone process leadiâg to the production of somatic
Vol . 15, No . 12
Poatreplication Repair of DNA in Naa®alian Cells
olutationa and cancer (see for example (84)) .
201 5
The findings that some XP cells
are normal in excision of dimes but have some kind of rbnormal poatreplication repair, leads further support to a relationship between this pracesa and the ultimate production of cancers . Kith rodent cell lines, there is evideac e for a link between some kind of caffeine-sensitive postreplication repair process and the production of chromosome aberrations in both Vicia faba and Chinese hamster cells (85) . Various work era have shown that caffeine increases the yield of mutations induced by UV or N-methyl N-nitroeourea (88-88), although others found a decreRSe (36) .
The interpretation of these results is ae yet not clear, but
a possible link between poatreplication repair and mutation induction is eug~ Bested by the findings that caffeine affects both these processes . References 1. 2. 3. 4, 5. 8. 7. 8. 9. 10 . 11 . 12 . ?3 . 14, 15 . 18 . 17 . 18 .
19 . 20 . 21, 22 . 23 . 24 .
8 . BTRAUSS, Life Sciences, in press . 8 . COOK, Photophyaiol . 5 191-233 (1970) . D . RSGAN, J . E . TROSKO and lI . L . CARRISR, Biophya . J . _8 319-325 (1988) . D . RUPP a~ P . Hq/ARD-FIAI~SRS, J . llol . Biol . 31 291-304 (1988) . 228 117-128 (1971) . N . IYSR and w. D . RUPP, Hiochim . Biophys, Acts HOMARD- FIANDSAS, B . D . RUPP, H . Y . IIILKIN3 ami R . 8 . COLS, Cold Spring Harbor 8yolp . Quant . Hiol . 33 196-207 (1988) . K . C . SMITH and D . H . C . 1LBiJN, J . Yol . Biol . 51 459-472 (1970) . H . A . BR II)GE8 end 8 . G . SEOGRICK, J . Hact . _117 1077-1081 (1974) . w, D . RUPP, C . S . 1fIIDS III, D . L . RENO and P . HOMARD-FLAI~SàB, J . Yol . Hiol . _81 25-44 (1971) . R . D . LEY, Photochem . Photobiol . 18 87-95 (1973) . A, K . GANSSAN, J . Mol . Hiol . i n press . J . A . HUBSAYAN a~ A, D . RIGGB, J . Yol . Biol . _32 327-341 (1988) . H . wSINTRAUH ; Nature New Hiol . 23E 195-197 (1972) . A . S . 11EYN, D . L . VIZARD, R . R . HSIIITT and R . Y . HUPPBÜSY, Photochem . Photobiol . in press . A . R . LBS1fANN earl S . STbY8r18, unpublished observations . R . A . YcGRATH and R . 11 . IIILLIAYB, Nature 212 534-535 (1988) . J . S . CLSpVIQt and G . S . THOIfAS, Biochem . Hiophye . Res . Comm . 3 8 203-208 (1989) . R . D . RUPP, E . ZIPSSR, C . V(N SSBSPi, D . RENO, L . PR08NITZ end P . HUIARDFIAl~ERB, In Time a~ Dose Relationship in Radiation Biology as Applied to Radiotherapy, Hrookhaven Natl . Lab . Publication BNL 50203 (c-57) 1-13 (1989) . R . S . YEYN and R . Y . HUllpFIItEY, Biophys . J . _11 295-301 (1971) . Y. FUJIIIARA and T . KQ~iDO, Hiochem, Biophys . Rea . Coa . 47 557-584 (1972) . A . R . LSBYANN, J . Mol . Hiol . 88 319-337 (1972) . Y . FUJIwARA, Sxpl . Cell Bee . 75 483-489 (1972) . 8 . N . HUHL, R . Y . STILLPAN, R . H . BSTLO* and J . D . RS(1AN, Hiophye . J . i2 1183-1191 (1972) . J . S . CLSAVSR, Proc . Nat . Aced . 8c1 . 83 428-435 (1989) .
H. J. J. A. V. P.
2016 25 . 28 . 27 . 28 . 29 . 30 . 31, 32 . 33 . 34 . 35 . 38 . 37 . 38 . 39 . 40 . 41 . 42, 43 . 44 . 45 . 48 . 47 . 48 . 49 . 50 . 51, 52 . 53 . 54 . 55 . 58 . 57, 58 . 59 . 80 . 81 . 82, 83 . 84 . 85 . 88 . 87, 88 .
Postreplication Repair of DNA in Mammalian Cells
S. B. H. S, A. A. H. A.
Vol . 15, No . 12
V . ZIPBSR, Ph .D thesis, Yale Univ . (1973) . N . BURL, R . H . BETL()II and J . D . R1~(3AN, personal communication, Y . SUTSEIIL4ND, Nature 248 109-112 (1974) . Y . F . CSIU and A . !! . RAUTH, Biochim . Hiophye, Acta 259 184-174 (1972) . R . LBB144NN end M . G . ORMB
13
Pergamon Press
2005-2016
MINIREVISiO P081RSPLICATION R1~AIR OF DNA IN 1fA18fALIAN CBLLB Alen R .
nnn
T
Y .R .C . Cell Mutation Unit, University of 8useex, Falmer, Brighton HN1 94G, Bagland
Ys®alien calls possess vecheniema for rmoving or circumventing various lesions is their DNA, and this has been the eub~ect of a review in this aeries by 8trause (1) .
Many lesions remain in the DNA in damaged cells, without pre Poetreplication repair of DNA may be
venting replication irov taking place .
defined is its broadest sense ae the manner is which the DNA replication machinery copes with lesions in the parental DNA strands .
Ii damaged bases
are copied incorrectly, this may lead to cell death, mutation and possibly carcinogeneeie .
The process ie therefore of great importance .
It will be die-
cussed principally with regard to davege ceased by ultraviolet (W) irradiation . Strictly speaking it ie not a repair process, since the lounge ae such ie not removed .
It ie closely associated with DNA replication .
Bacteria can remove W-induced cyclobutane pyrimidine divers cram DNA (a) by photoreactivation, a light-induced splitting of pyrimidine diniere in situ , which has not been detected in vivo is placental eiammale (reviewed in Ref . (2)), ar (b) by excision repair, which oleo occurs in human cells (3) but only to a very limited extent in rodent cells .
Despite this poor ability of marry mamvslian
cells to remove pyrimidine divers, they can in general survive W flumcee which produce 105 -108 such lesions per cell .
Poetreplication Repair in Bacteria In strains of Becherichia coli unable to excise pyrividine diners Rupp and 2005
2006
Postreplication Repair of DNA in Mammalian Cells
Vol. 15, No . 12
Howard-Flanders ßcund that newly-synthesized DNA in W-irradiated cells was initially oß lower molecular weight than is unirradiated cells (4) .
This
molecular weight oß the DNA gradually increased on subsequent incubation, eventually reaching the same size as that of DNA from unirradiated controls . They postulated that the DNA "replicaee" was temporarily delayed at each dimes (to account ßor the W-induced inhibition oß DNA synthesis) and then continued beyond the dieter, leaving a gap .
This gap was subsequently sealed .
In later
work, they showed that Dd~W replicated in W-irradiated cells did indeed contain single-stranded regions, about 1000 nucleotides in length (5) .
ibccellent
indirect evidence has been obtained that these single-stranded regions are the result oß gaps opposite the dieters (B-S) .
The gap-Billing is achieved by a
process involving mates-strand recombinational exchanges oß large amounts oß DNA (9, 10) and recent evidence indicates that the dieters (ae measured by sites susceptible to the W-specißic T4 endonucleaee-V) become equally distributed between parental and daughter strands (11) . In animal cells, a similar process seems to occur, marked difßer~cde in details.
although there are some
The writing oß this review comes at a time when
some oß the interpretations oß the available data ere being questioned, so careßu1 attempts will be made to distinguish ßacta and experimental results, ßrom interpretation and speculation .
Interpretations presented will naturally
reflect my personal bias, and should therefore be treated with appropriate caut ion.
Ilechaniema oß DNA Replication in Damaged Cells D3QA in mammalian chromoe~ea is synthesized by many independent replicating forks.
Replication occurs b1d1ractione1ly (12,13) ßrom multiple origins at a
rate oß 0" 5-2 " 5 N/min per replication Bark . origins vary ßrom 16 to 200 microns .
Setimatee oß the distance between
After replication adjacent replication
segments (replicona) are joined together to produce very long DNA strands . As with bacteria, it has been shown in many mammalian cell lines, that W
Vol. 15, No . 12
Poatreplicatioa Repair of DNA is Mammalian Cells
and other insults decrease the rate of
DTiA
replicatirn .
This is presumably
a consequence of some or all of the lesions in the changed DNA
replication machinery .
200 7
Dd~iA
delaying the
The fraction of pyrimidine diners in
DiNA
repli-
cated after W-irradiation of Chinese hamster cells ie the same as that in unreplicated
DNA,
indicating that the dLere only delay
DNA
replication tem-
porarily and do not completely block it (14,16) . Various hypothetical schwas by which lesions in growth of daughter-strands are shown in Fig.l .
DNA
could affect the
Hyntheeis may continue r oued
the lesion (Fig . ld) possibly after a delay, it may be halted completely (Fig . 1a),
it may be delayed and then continue beyond the lesion leaving a
discontinuity opposite the lesion (Fig .
Ib) .
Thin say be sealed some time
later ae is Fig . Ib or very shortly after its formation (Fig . lc ) . FIO. 1
O
b
o
.
. . "n
..
0
C
d
O
O
O
0 o u
00
0
.
o0
0 0
o
a
"o
O
Ct
.
Fig. 1 . Yodels for replication on damaged templates ; a - lesions completely block replication ; b - gape left opposite lesions and filled in ease time later; c - as b, but gape filled in rRpidly; d - continuous synthesis peat lesions . 0, origins of replication ; ~, lesions ; ~, ü lled in gap ; -, parental DNA ; ~, newly-synthesized DNA .
Bize of
DNA
Made is W-Irradiated Cells
Yo st of the work on poetreplication repair in mammalien celle hoe involved meas~emeata of the mol . wt, of DNA from lysed celle, using the technique of centrifugation in alkaline sucrose gradients (18) .
The study of
D1~iA
of mol .
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Postreplication Repair of DNA in Mammalian Cells
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wt . greater than 5 x 108 ie fraught with dißßiailtiee caused by incomplete denaturatioa, and aggregation and entanglement effects, whidi are little understood .
In order to overcome these problems, steps have usually been taken to
fragment the DNA to mol . wts . below 5 x 10 8 immediately before centrifugation . This has been achieved by prolonged lyeie treatments in alkali, or by low doses oß X- or Y-irradiation . Sxperimente by many workers, using alkaline sucrose gradients,
have shown
that is several Chinese hamster and mouse cell lines, as in bacteria, newlysynthesized DNA (labelled by a pulse oß 3H-thymidine) in W-irradiated cells is smaller than that in unirradiated cells (17-22) .
8lmilar observations have
been made with normal human cells, and cells derived from patients carrying the inherited akin disease xeroderma pigmentoeum (XP) (23), a disorder characterized by a deficiency in the ability oß the cells to excise W-induced pyrimidine diniere from their DNA (24 ). ßluences oß 7-100
Jm 2,
The above experim~te were carried auf using W
and were interpreted on the bacterial model, ae evidence
far discontinuities in the daughter-strands opposite pyrimidine diniere (Fig . lb). In order to eliminate the possibility that the results are merely due to the slower rate oß synthesis in W-irradiated cells several oß the above walkers pulse-labelled irradiated cells ßas longer times than the unirradiated controls, so that the same amount oß radioactivity was incorporated in unirradiated and irradiated cells. Some evidence hoe been obtained for single-stranded regions in replicated DNA from W-irradiated sauee cells (25), similar to those.ßound in S.ooli (6). The size oß short pieces oß newly-synthesized i~iA is approximately equal to the average distance between dimes in the parental strands, as calculated from the fraction oß thymine in pyrimidine diniere (21,23,25) .
Thin ie caaeietent with
the idea that the gaps in the new DNA are opposite the diniere.
It should, how-
ever, be pointed out that both the above parauetere are eub~ect to large experimental errors and that this rather crude comparison ie the only evidence available to indicate that the gape are in fact opposite diniere in placental mammals .
Postreplication Repair of DNA in Mammalian Cells
Vol . 15, No . 12
200 9
Recently, Buhl, Setlow and Regan using a marsupial cell line, compared the size of newly-synthesized DNA in cells which had been W-irradiated to that from cells which were W-irradiated followed by photoreactivation to remove some of the diniere (28) .
In the latter case the DNA was larger than in the former sug~
Besting again that the gape wpa a opposite the dimes e .
Although photoreacti-
vesting enzyme has recently been detected in human leucocytee (27), photoreactivat ion in vivo has never been detected in placental mammals .
Hence this type
of experiment can only be carried out with marsupials and lower orders . In cotrtraet to the results discussed eo far, Chiu and Rauth found that in W-irradiated souse L cells, newly-synthesized DNA was the same size se that in unirradiated cells (28), provided that care was taken to eliminate labelling artefacts (29), by labelling equivalent lengths of DNA in irradiated and unirradiated cells .
DS~A made in cells irradiated with high W iluencea
(i 20 Jm2) was smaller than that in unirradiated cella, but the size of the pieces synthesized was cvneiderably greater than the inter-dimec~distance . These data suggested that replication continued round the dieter (Fig . id) but could also be explained if gape were left sad sealed vea~y rapidly (Fig . 1c ) . In later work Rauth et al ., compered the mol . wts . of DNA synthesized in Wirradiated L cells, CHO and HeLa cells (30) . the cell lines .
They found differences between
These could be explained by differing raYres of gap-filling in
these cell lines, or by the presence of both a discontinuous synthesis (with gape) and a continuous synthesis peat dieters . At the opposite extreue Edenberg has recently suggested that the low mol . wt . DANA seen in alkaline sucrose gradients does not represent DNA strands coatsining small gape, but that synthesis is halted far a long period at the dimmers rather than continuing beyond after a short delay (31) .
In my opinim ,
presently available data do not substantiate this suggestion (32) and far the rest of this review it will be aesuved that discontinuities are in fact left opposite dieters, end then sealed (as in Fig . Ib or Ic) although this remains to be rigorously proven .
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Poetreplication Repair of DNA in Mammalian Cells
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Filling in of Rape The low molecular weight DNA in UV-irradiated cells is detected by alkaline sucrose sedimentation of DNA from cells labelled with a short pulse of 3H-thymidine .
Ii this pulse ie followed by a chase in unlabelled thymidine,
it has been sound in all the cell Bass examined that the low mol . wt . DNA increases
3a size, eventually attaining a mol . wt . similar to that from
uairradiated cells (17-19,21-23) .
This increase in mol . wt . i e the result of
two processes : (a)
those associated with normal DNA replication and therefore seen eleo in
unirradiated cells (chain elongation within replicating units and joining of adjacent replicating units,
(b)
filling-in of daughter-strand gape in damaged celle .
Gap-filling is
inhibited by the DNA synthesis inhibitors hydro~urea (21,23,33), cytosine arabinoeide and excess thymidine (33), and by
actinomycin D (33), but not by
cycloheximide, as inhibitor of protein synthesis (33,34) .
In rodent cells it
ie possible to inhibit gap-filling without greatly sirecting DNA replication, with caffeine (17, 20,22,35,25,38), or its analogue
theophylline (37) .
Althougà caffeine does affect normal DNA synthesis to a small extent (38) its effect on W-irradiated cells is far more pronounced .
In the latter case,
in the presence of caffeine, low mol . wt . DNA accumulates during e prolonged labelling period (25,38), wher~a in its absence it slowly increases in size due to filling~in of the gape .
In normal human cell linen caffeine has
hardly any inhibitory effect on gap-filling (22,39-41), although under some conditions a small inhibition may be observed (40,42) .
In XP cells, however,
it has a vea~y marked effect (see below) .
These effects of caüeine correlate
very well with effects on cell survival .
Caffeine sensitizes many rodent
cells to W-irradiation (43) but only if present during the DNA synthesis period (44), and it does not sensitize human (HeIa) cells (46) .
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Postreplication Repair of DNA in Mammalian Celle
201 1
Mechanism of gap-filling Ia S .coli Rupp et al ., (9) postulated that the daughter-strand gaps were filled by a process in which the genetic information lost by the formation of a gap opposite a dieter was retrieved by a sister-attend recombinational exchange . The piece of parental DNA from the sister duplex, which contained the eiesing sequence of nucleotides was inserted in the gap opposite the dieter .
The
resulting gap is the parental strand of the sister duplex, could then be filled in by repair replication, since it was opposite an intact template .
Hy tech-
niques involving density-labelling and equilibrium centrüugatioa in alkaline CeCl (9) or bromodeoxyuridine photolysie (10) evidence has been obtained for
end-to-mod association of par~tal and daughter-strand DI~iA after W irradiation indicating that exchanges of large lengths of DNA (about 6 x 108 daltons) between parental and daughter-strand had occurred . Attempts to detect exchanges in mammaliea cells have been made using photoZyais of bromodeoxyuridine-substituted DNA (21), alkaline CsCl c~trifugatioa (25,48), and detection of pyrimidine dieters in daughter-strand Dl~iA (47, 48) .
These techniques have all given positive indication of exchanges in
B .coli (9-i!), but Gave failed to detect such exchanges in mammalian cells .
Fhrthermore the daughter-strand gape seem to be sealed by de aovo synthesis with a stretch of DNA approximately 1000 nucleotides in length (21,49,48) . There is therefore at present no evidence to support the bacterial model of gap-filling by recombinational exchanges .
If such exchanges exist, they have
not beg detectable by the techniques available, and must be leas than about 100 nucleotides in length .
Certainly ~oae exchanges such as occur in S .coli
(9,10) can be ruled out .
Recovery_ of Ability to Synthesize High ![ol . i"t .- 7$iA at Ixte Times attar Irradiation All the walk reviewed so far has dealt with stretches of DNA synthesized within an hour or two after irradiation .
Hiace rodent cells have only a very low
Poatreplication Repair of DNA is Maa~alian Cells
2012
Vol . 15, No . 12
level of excision of pyrieidine dimere (50,37,36,14), it would be anticipated that all DNA synthesized within the Piret generation after W-irradiation would initially curtain gape, since the template still contained dimes . Surprisingly, however, it was shown in rodent cells that the DNA synthesized in a short pulse-label given 8 - 8 hours niter irradiation was the same size as that in unirradiated cells, i .e ., no gape could be detected (19,37) .
This
recovery of the ability to synthesize high mol . wt . DNA did not occur in a W sensitive Chinese hamster cell line, B14 (19) .
It occurred in both normal
human cells and in the excision-deficient XP cell lines at the same rate (81) . F1u~thermore, it took place even if DNA synthesis was prevented by hydroxyurea between the ties of irradiation end pulse-labelling (81) . ie a mystery .
This phenomenon
1~Scplenatione which have been put forward include :
(1)
rapid gap-Pilling at late times after irradiation (37),
(2)
the possibility that the gaps are opposite soee lesion other than pyrimidine dimere, and this lesion is excised is the First Pew hours after irradiation in all the cell lines studied (51),
(3)
a coniarmational change of the dimere such that they no longer lead to the production oP daughter-strand gaps (81) .
None of these explanations satisfactorily account Por all the available data, sad the phenomenon remains a mystery though its existence ie certainly beyond doubt .
Gap-filling in Xeroderma Pig tosum Çelle It has recently been shown that 7CP cells can be divided into at lee at five or six complementation groups on the basis oP cell Fusion experiments (82,53) . In at least three of these, gape were left in daughter-strand DNA after Wirradiation and these gape were subsequently sealed (23) .
In as XP line from
the "classical XP" complementation group both the gap-Pilling and the recovery of the ability to synthesize high mol . wt . DNA at late times after irradiation were inhibited by caffeine (39) .
Normal cells were unaffected .
Other XP
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Postreplication Repair of DNA is ilaamalian Cells
2013
cosplementatica groups were not examined for caffeine sensitivity. One group o! patients have been clinically diagnosed ae having 7[P, but unlike moat of the other cases eaa
In fibro-
blast cells from three of these cases the ooavereion of low mol . wt . DNA to high ool . wt . after W-irradiation was considerably slower than in normal fibroblaeta (40) .
Furthermore this coavea~eian was drastically inhibited by
caffeine, which had very little effect on normal cells .
A "classical 7[P"
line and a line with the de-8aactis~accione syndrome showed intermediate behaviour both with regard to rate of gap-filling and inhibition by caffeine (40) .
Thus the 7[P lines normal in excision-repair have same deficiency in
postrepücation-repair, although the nat~ae of this deficiency is at present not clear .
From these ündings we have the interesting and rather surprising
observation that a deficiency in postreplication repair and various defects in excision-repair at the cellular level seem to give rise to similar clinical symptoms in man .
It seems that pyrimidine
dimere induced is human skin by
sunlight (66) are highly likely to lead to skin cancer either if the dimere are not excised (as in most XP cases) or if they are replicated in a defective manner (as in the varisate discussed above) . Poetreplicatian Repair in Cells Damaged with Ch®kale The way in which the Dd1A replication machineay copes with lesions induced by chemicals such as alkylating agents end other mutegens, has not as yet been studied in ae much detail as with W-irradiation .
Methyl methane eulphonate
(1018) ie an alkylating agent which produces a variety of lesions in D~ (68) . DNA synthesized in the first few hours after treatment with 1W8 ie abnormally short (57,68) and ae with W these abort chAine are subsequently elongated. Using
HND-cellulose chromatography to detect single-strand regions of DNA,
8cudiero and 8traues found a higher proportion of such regions in 1018-treated
Postreplication Repair of DNA in Mammalian Cells
201 4
cells than in untr~ted controls (59) . lesions blocks DNA synthesis .
Vol . 15, No . 12
They suggest that one of the induced
After removal of the lesion, replication can
continue . Based on detailed kinetic atudiea of the effects of N-methyl N-nitrosourea and sulphur mustard on DNA synthesis, Roberta and colleagues inferred that lesions i~uced by these ag~ta somehow interfere with replication, but they can be overcome poatreplicatively (60) .
Ae with W-irradiation caffeine sensi-
tized Chinese hamster cells but not HeIa cells to alkylating agents and similarly affected DNA synthesis and cell progression in the rodent but not the human line .
Preliminary molecular atudiea am the size of DNA synthesized in
Chinese hamster cells treated with N-methyl N-nitrosourea conürmed that the induced lesions acmehow interfered with the normal synthesis of high mol, wt . DNA and caffeine had a further inhibitory effect (81) .
Similarly Yan den
Berg found that treatment of HeLa cells with the carcinogen methylazoxy methanol acetate interfered with chain elongation (82) .
Finally Troeko et al .
found that in Chinese hamster cells treated with N-acetoxyaminoiluorene,se with W , caffeine inhibited the eventual synthesis of him mol . wt . DNA (83) . Thus in cells treated with alkylating agents, ae with W , there ae®s to be a caffeine-sensitive S-phase specific poatreplicative repair process .
The
mechanism seems to have acme eimiliaritiea to the dimer/gap mechani® for poatreplication repair in W-irradiated cells, but may düfer significantly in details .
Only preliminary data is available at present .
Relationship of Poetreplication Repair to Mutation, Chromosome Aberratim e and 0arçinogenesie In cells from patients with "classical XP" or the de-8anctie~accione eyndroue pyrimidine dimes are not excised, and more of these lesions moat be "repaired" poatreplicatively than in normal cells .
Since patients with XP inevitably
develop akin carcinomas, it has been speculatively suggested that postreplication repair is an error-prone process leadiâg to the production of somatic
Vol . 15, No . 12
Poatreplication Repair of DNA in Naa®alian Cells
olutationa and cancer (see for example (84)) .
201 5
The findings that some XP cells
are normal in excision of dimes but have some kind of rbnormal poatreplication repair, leads further support to a relationship between this pracesa and the ultimate production of cancers . Kith rodent cell lines, there is evideac e for a link between some kind of caffeine-sensitive postreplication repair process and the production of chromosome aberrations in both Vicia faba and Chinese hamster cells (85) . Various work era have shown that caffeine increases the yield of mutations induced by UV or N-methyl N-nitroeourea (88-88), although others found a decreRSe (36) .
The interpretation of these results is ae yet not clear, but
a possible link between poatreplication repair and mutation induction is eug~ Bested by the findings that caffeine affects both these processes . References 1. 2. 3. 4, 5. 8. 7. 8. 9. 10 . 11 . 12 . ?3 . 14, 15 . 18 . 17 . 18 .
19 . 20 . 21, 22 . 23 . 24 .
8 . BTRAUSS, Life Sciences, in press . 8 . COOK, Photophyaiol . 5 191-233 (1970) . D . RSGAN, J . E . TROSKO and lI . L . CARRISR, Biophya . J . _8 319-325 (1988) . D . RUPP a~ P . Hq/ARD-FIAI~SRS, J . llol . Biol . 31 291-304 (1988) . 228 117-128 (1971) . N . IYSR and w. D . RUPP, Hiochim . Biophys, Acts HOMARD- FIANDSAS, B . D . RUPP, H . Y . IIILKIN3 ami R . 8 . COLS, Cold Spring Harbor 8yolp . Quant . Hiol . 33 196-207 (1988) . K . C . SMITH and D . H . C . 1LBiJN, J . Yol . Biol . 51 459-472 (1970) . H . A . BR II)GE8 end 8 . G . SEOGRICK, J . Hact . _117 1077-1081 (1974) . w, D . RUPP, C . S . 1fIIDS III, D . L . RENO and P . HOMARD-FLAI~SàB, J . Yol . Hiol . _81 25-44 (1971) . R . D . LEY, Photochem . Photobiol . 18 87-95 (1973) . A, K . GANSSAN, J . Mol . Hiol . i n press . J . A . HUBSAYAN a~ A, D . RIGGB, J . Yol . Biol . _32 327-341 (1988) . H . wSINTRAUH ; Nature New Hiol . 23E 195-197 (1972) . A . S . 11EYN, D . L . VIZARD, R . R . HSIIITT and R . Y . HUPPBÜSY, Photochem . Photobiol . in press . A . R . LBS1fANN earl S . STbY8r18, unpublished observations . R . A . YcGRATH and R . 11 . IIILLIAYB, Nature 212 534-535 (1988) . J . S . CLSpVIQt and G . S . THOIfAS, Biochem . Hiophye . Res . Comm . 3 8 203-208 (1989) . R . D . RUPP, E . ZIPSSR, C . V(N SSBSPi, D . RENO, L . PR08NITZ end P . HUIARDFIAl~ERB, In Time a~ Dose Relationship in Radiation Biology as Applied to Radiotherapy, Hrookhaven Natl . Lab . Publication BNL 50203 (c-57) 1-13 (1989) . R . S . YEYN and R . Y . HUllpFIItEY, Biophys . J . _11 295-301 (1971) . Y. FUJIIIARA and T . KQ~iDO, Hiochem, Biophys . Rea . Coa . 47 557-584 (1972) . A . R . LSBYANN, J . Mol . Hiol . 88 319-337 (1972) . Y . FUJIwARA, Sxpl . Cell Bee . 75 483-489 (1972) . 8 . N . HUHL, R . Y . STILLPAN, R . H . BSTLO* and J . D . RS(1AN, Hiophye . J . i2 1183-1191 (1972) . J . S . CLSAVSR, Proc . Nat . Aced . 8c1 . 83 428-435 (1989) .
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S. B. H. S, A. A. H. A.
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V . ZIPBSR, Ph .D thesis, Yale Univ . (1973) . N . BURL, R . H . BETL()II and J . D . R1~(3AN, personal communication, Y . SUTSEIIL4ND, Nature 248 109-112 (1974) . Y . F . CSIU and A . !! . RAUTH, Biochim . Hiophye, Acta 259 184-174 (1972) . R . LBB144NN end M . G . ORMB
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