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Finally, Polyubiquitinated PCNA Gets Recognized
Molecular Cell
Previews Finally, Polyubiquitinated PCNA Gets Recognized Michelle K. Zeman1 and Karlene A. Cimprich1,* 1Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2012.07.024
Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, together with Weston et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA and plays an important role in restarting stalled replication forks. DNA damage presents a challenge to genome integrity during all cell-cycle phases, but lesions encountered during DNA replication can be particularly problematic. These lesions stall the replication fork, leading to unstable structures prone to rearrangement and mutation (Figure 1A) (Branzei and Foiani, 2010). In order to prevent this, cells have evolved ways of stabilizing the stalled fork and promoting the resumption of DNA replication. The DNA damage tolerance (DDT) pathway is a key contributor to this process, orchestrating lesion bypass through posttranslational modification of the replicative clamp, PCNA. Intriguingly, it has been known for many years that polyubiquitination of PCNA with a K63-linked chain signals an error-free form of lesion bypass via template switching (Ulrich and Walden, 2010). However, the exact function of the polyubiquitinated PCNA, and the mechanism behind this form of lesion bypass, has long been a mystery. This month, three papers—from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, and from Weston et al. (2012) in Genes and Development— characterize new biochemical activities and substrates of ZRANB3/AH2, which have significant implications for the role of PCNA polyubiquitination and for the molecular mechanism behind template switching. Although ZRANB3/AH2 has been previously described as an annealing helicase or translocase capable of ‘‘rewinding’’ denatured single-stranded DNA (ssDNA) in vitro (Yusufzai and Kadonaga, 2010), little was known about its roles in vivo. Collectively, the current studies show that ZRANB3/AH2 is recruited to sites of DNA damage through an interaction with PCNA, in order to promote fork restart after fork stalling. This recruitment is
mediated by three domains. Two are required for direct interaction with PCNA: a conserved PCNA-interacting protein (PIP) box, and a C-terminal AlkB2 PCNAinteraction motif (APIM). The third is an NPL4 zinc finger (NZF), a specialized type of ubiquitin-binding domain which can specifically recognize K63-linked ubiquitin chains. This is one of the most interesting findings, as Ciccia et al. show that this NZF motif is required for a specific interaction with the K63-linked polyubiquitinated form of PCNA in vitro and for retention of ZRANB3 at damage sites in vivo. They also show this association has functional consequences, as these motifs are required for efficient fork restart in cells. Both the current and previous work suggests multiple ways by which ZRANB3/AH2 might act to promote fork restart. Its ability to reanneal ssDNA ‘‘bubbles’’ has been speculated to regulate the balance between wound and unwound parental DNA at a stalled fork. This type of activity could oppose the replicative helicase and other unwinding activities to stabilize the fork structure and minimize the accumulation of ssDNA (Driscoll and Cimprich, 2009). Interestingly, however, Ciccia et al. also report that ZRANB3/AH2 exhibits translocase activity on two additional substrates, a finding which could have implications for fork restart. First, ZRANB3/AH2 can regress stalled forks, which could facilitate lesion bypass by providing access to the newly replicated sister chromatid. This would allow the cell to avoid the damaged DNA entirely by using the undamaged chromatid as a template (Figure 1B). Given the specificity of ZRANB3/ AH2 for binding polyubiquitinated PCNA, a critical signal for template switching at stalled forks, it is exciting to postulate
that fork regression may be triggered by recruitment of ZRANB3/AH2 to this modification. Second, Ciccia et al. show that ZRANB3/AH2 can disrupt D-loop structures in vitro. This raises the possibility that ZRANB3/AH2 prevents unnecessary recombination events by dissolving inappropriate D loops at the stalled replication fork or possibly at gaps left behind the fork (Figure 1C). Consistent with this idea, ZRANB3/AH2 is shown to suppress sister-chromatid exchanges, common crossover events during perturbed replication (Ciccia et al., 2012). While sisterchromatid exchanges are not deleterious to the cell per se, a higher rate of D-loop formation increases the likelihood of inaccurate strand invasion and, by extension, the chance for alteration of genetic information. Surprisingly, the paper from Weston et al. (2012) also reveals a novel function for ZRANB3/AH2 as a structure-specific endonuclease. The ability of ZRANB3/ AH2 to cut replication fork structures in vitro relies on its HNH motif, a functionally divergent domain found in a variety of DNA-binding proteins. The authors suggest that this endonuclease activity, in conjunction with fork regression, may contribute to the removal of DNA lesions (Figure 1D). As such a model involves the repair of DNA damage at the fork, rather than lesion bypass, this finding could suggest that PCNA polyubiquitination plays a role in replication-associated DNA repair as well as DDT. ZRANB3/AH2 is the second annealing helicase to be characterized, following SMARCAL1/HARP (Driscoll and Cimprich, 2009), and the work of Yuan et al. (2012) suggests that there are at least two more members of this family, Rad54L and SMARCA1. All four proteins contain a HARP-like (HPL) domain, which
Molecular Cell 47, August 10, 2012 ª2012 Elsevier Inc. 333
Molecular Cell
Previews
Z
was originally found to confer or if its biochemical pro▼ annealing helicase activity perties are modulated differto SMARCAL1/HARP (Ghosal ently in cells. Clearly, howAn et al., 2011). In addition, ever, these studies open Nicked Fork e n s a a e le ty translocases from other famimany new avenues of invesB Regressed Fork ctivityaling uc vi N acti Lesion bypass lies, such as HLTF and tigation by linking PCNA Fork restart FANCM, are also capable of polyubiquitination to specific regressing forks (Unk et al., biochemical activities and of 2010). This raises the quesby beginning to address the n Regressed Fork io pt ops tions: Why does the cell long-standing question of u r A Stalled Fork is lo D Dneed such a variety of seemwhat recognizes polyubiquitiingly redundant players? nated PCNA. Could these translocases be working in a damage- or sequence-specific manner? REFERENCES C Recombination-Mediated D Repaired Fork In different DNA compartTemplate Switching Lesion repair Sister chromatid exchange Fork restart Branzei, D., and Foiani, M. (2010). ments? With different molecNat. Rev. Mol. Cell Biol. 11, ular partners? At least in the 208–219. Figure 1. Fork Restart Activities of ZRANB3/AH2 case of SMARCAL1/HARP (A) In the presence of DNA damage (red star), the replication fork stalls, allowCiccia, A., Nimonkar, A.V., Hu, Y., and ZRANB3/AH2, it seems ing for the accumulation of RPA (brown) on single-stranded DNA. PCNA Hajdu, I., Achar, Y.J., Izhar, L., clear that these are not (purple) is polyubiquitinated by proteins in the DNA damage tolerance Petit, S.A., Adamson, B., Yoon, pathway to induce template switching. According to the current work, polyuJ.C., Kowalczykowski, S.C., et al. redundant proteins. The (2012). Mol. Cell 47, this issue, biquitinated PCNA is recognized by ZRANB3/AH2 (green), which may remodel mechanisms and kinetics of 396–409. the fork and promote fork restart in several ways. recruitment to stalled forks (B) ZRANB3/AH2 demonstrates fork regression activity in vitro, which may Driscoll, R., and Cimprich, K.A. facilitate template switching and lesion bypass in cells. are distinct, and they are (2009). Genes Dev. 23, 2359–2365. (C) ZRANB3/AH2 is capable of disrupting D-loop structures in vitro, an activity not functionally epistatic in which may prevent sister chromatid exchanges in vivo. Ghosal, G., Yuan, J., and Chen, J. several assays (Ciccia et al., (D) ZRANB3/AH2 exhibits structure-specific endonuclease activity on the (2011). EMBO Rep. 12, 574–580. leading strand of replication fork structures in vitro (green triangle), which, in 2012, Yuan et al., 2012). conjunction with fork regression, may lead to repair of the lesion at the stalled Finally, although ZRANB3/ Ulrich, H.D., and Walden, H. (2010). replication fork. Nat. Rev. Mol. Cell Biol. 11, AH2 exhibits a specific pref479–489. erence for K63-polyubiquitinated PCNA, it is not clear whether ubiquitinate PCNA (Unk et al., 2010). Unk, I., Hajdu´, I., Blastya´k, A., and Haracska, L. ZRANB3/AH2 is functioning as a new What, then, is the role of ZRANB3/AH2? (2010). DNA Repair (Amst.) 9, 257–267. component in the DDT pathway, in a This may be clarified through DNA muta- Weston, R., Peeters, H., and Ahel, D. (2012). Genes parallel DNA repair pathway, or with tion analysis and epistasis studies with Dev. 15, 1558–1572. something else entirely. HLTF, a key ubiq- other DDT proteins. Electron microscopy Yuan, J., Ghosal, G., and Chen, J. (2012). Mol. Cell uitin ligase and translocase in the DDT and physical interaction studies may 47, this issue, 410–421. pathway, also has fork regression activity, also help reveal if ZRANB3/AH2 can Yusufzai, T., and Kadonaga, J.T. (2010). Proc. Natl. is important for fork restart, and can poly- actively promote fork regression in vivo, Acad. Sci. USA 107, 20970–20973.
334 Molecular Cell 47, August 10, 2012 ª2012 Elsevier Inc.
Previews Finally, Polyubiquitinated PCNA Gets Recognized Michelle K. Zeman1 and Karlene A. Cimprich1,* 1Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2012.07.024
Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, together with Weston et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA and plays an important role in restarting stalled replication forks. DNA damage presents a challenge to genome integrity during all cell-cycle phases, but lesions encountered during DNA replication can be particularly problematic. These lesions stall the replication fork, leading to unstable structures prone to rearrangement and mutation (Figure 1A) (Branzei and Foiani, 2010). In order to prevent this, cells have evolved ways of stabilizing the stalled fork and promoting the resumption of DNA replication. The DNA damage tolerance (DDT) pathway is a key contributor to this process, orchestrating lesion bypass through posttranslational modification of the replicative clamp, PCNA. Intriguingly, it has been known for many years that polyubiquitination of PCNA with a K63-linked chain signals an error-free form of lesion bypass via template switching (Ulrich and Walden, 2010). However, the exact function of the polyubiquitinated PCNA, and the mechanism behind this form of lesion bypass, has long been a mystery. This month, three papers—from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, and from Weston et al. (2012) in Genes and Development— characterize new biochemical activities and substrates of ZRANB3/AH2, which have significant implications for the role of PCNA polyubiquitination and for the molecular mechanism behind template switching. Although ZRANB3/AH2 has been previously described as an annealing helicase or translocase capable of ‘‘rewinding’’ denatured single-stranded DNA (ssDNA) in vitro (Yusufzai and Kadonaga, 2010), little was known about its roles in vivo. Collectively, the current studies show that ZRANB3/AH2 is recruited to sites of DNA damage through an interaction with PCNA, in order to promote fork restart after fork stalling. This recruitment is
mediated by three domains. Two are required for direct interaction with PCNA: a conserved PCNA-interacting protein (PIP) box, and a C-terminal AlkB2 PCNAinteraction motif (APIM). The third is an NPL4 zinc finger (NZF), a specialized type of ubiquitin-binding domain which can specifically recognize K63-linked ubiquitin chains. This is one of the most interesting findings, as Ciccia et al. show that this NZF motif is required for a specific interaction with the K63-linked polyubiquitinated form of PCNA in vitro and for retention of ZRANB3 at damage sites in vivo. They also show this association has functional consequences, as these motifs are required for efficient fork restart in cells. Both the current and previous work suggests multiple ways by which ZRANB3/AH2 might act to promote fork restart. Its ability to reanneal ssDNA ‘‘bubbles’’ has been speculated to regulate the balance between wound and unwound parental DNA at a stalled fork. This type of activity could oppose the replicative helicase and other unwinding activities to stabilize the fork structure and minimize the accumulation of ssDNA (Driscoll and Cimprich, 2009). Interestingly, however, Ciccia et al. also report that ZRANB3/AH2 exhibits translocase activity on two additional substrates, a finding which could have implications for fork restart. First, ZRANB3/AH2 can regress stalled forks, which could facilitate lesion bypass by providing access to the newly replicated sister chromatid. This would allow the cell to avoid the damaged DNA entirely by using the undamaged chromatid as a template (Figure 1B). Given the specificity of ZRANB3/ AH2 for binding polyubiquitinated PCNA, a critical signal for template switching at stalled forks, it is exciting to postulate
that fork regression may be triggered by recruitment of ZRANB3/AH2 to this modification. Second, Ciccia et al. show that ZRANB3/AH2 can disrupt D-loop structures in vitro. This raises the possibility that ZRANB3/AH2 prevents unnecessary recombination events by dissolving inappropriate D loops at the stalled replication fork or possibly at gaps left behind the fork (Figure 1C). Consistent with this idea, ZRANB3/AH2 is shown to suppress sister-chromatid exchanges, common crossover events during perturbed replication (Ciccia et al., 2012). While sisterchromatid exchanges are not deleterious to the cell per se, a higher rate of D-loop formation increases the likelihood of inaccurate strand invasion and, by extension, the chance for alteration of genetic information. Surprisingly, the paper from Weston et al. (2012) also reveals a novel function for ZRANB3/AH2 as a structure-specific endonuclease. The ability of ZRANB3/ AH2 to cut replication fork structures in vitro relies on its HNH motif, a functionally divergent domain found in a variety of DNA-binding proteins. The authors suggest that this endonuclease activity, in conjunction with fork regression, may contribute to the removal of DNA lesions (Figure 1D). As such a model involves the repair of DNA damage at the fork, rather than lesion bypass, this finding could suggest that PCNA polyubiquitination plays a role in replication-associated DNA repair as well as DDT. ZRANB3/AH2 is the second annealing helicase to be characterized, following SMARCAL1/HARP (Driscoll and Cimprich, 2009), and the work of Yuan et al. (2012) suggests that there are at least two more members of this family, Rad54L and SMARCA1. All four proteins contain a HARP-like (HPL) domain, which
Molecular Cell 47, August 10, 2012 ª2012 Elsevier Inc. 333
Molecular Cell
Previews
Z
was originally found to confer or if its biochemical pro▼ annealing helicase activity perties are modulated differto SMARCAL1/HARP (Ghosal ently in cells. Clearly, howAn et al., 2011). In addition, ever, these studies open Nicked Fork e n s a a e le ty translocases from other famimany new avenues of invesB Regressed Fork ctivityaling uc vi N acti Lesion bypass lies, such as HLTF and tigation by linking PCNA Fork restart FANCM, are also capable of polyubiquitination to specific regressing forks (Unk et al., biochemical activities and of 2010). This raises the quesby beginning to address the n Regressed Fork io pt ops tions: Why does the cell long-standing question of u r A Stalled Fork is lo D Dneed such a variety of seemwhat recognizes polyubiquitiingly redundant players? nated PCNA. Could these translocases be working in a damage- or sequence-specific manner? REFERENCES C Recombination-Mediated D Repaired Fork In different DNA compartTemplate Switching Lesion repair Sister chromatid exchange Fork restart Branzei, D., and Foiani, M. (2010). ments? With different molecNat. Rev. Mol. Cell Biol. 11, ular partners? At least in the 208–219. Figure 1. Fork Restart Activities of ZRANB3/AH2 case of SMARCAL1/HARP (A) In the presence of DNA damage (red star), the replication fork stalls, allowCiccia, A., Nimonkar, A.V., Hu, Y., and ZRANB3/AH2, it seems ing for the accumulation of RPA (brown) on single-stranded DNA. PCNA Hajdu, I., Achar, Y.J., Izhar, L., clear that these are not (purple) is polyubiquitinated by proteins in the DNA damage tolerance Petit, S.A., Adamson, B., Yoon, pathway to induce template switching. According to the current work, polyuJ.C., Kowalczykowski, S.C., et al. redundant proteins. The (2012). Mol. Cell 47, this issue, biquitinated PCNA is recognized by ZRANB3/AH2 (green), which may remodel mechanisms and kinetics of 396–409. the fork and promote fork restart in several ways. recruitment to stalled forks (B) ZRANB3/AH2 demonstrates fork regression activity in vitro, which may Driscoll, R., and Cimprich, K.A. facilitate template switching and lesion bypass in cells. are distinct, and they are (2009). Genes Dev. 23, 2359–2365. (C) ZRANB3/AH2 is capable of disrupting D-loop structures in vitro, an activity not functionally epistatic in which may prevent sister chromatid exchanges in vivo. Ghosal, G., Yuan, J., and Chen, J. several assays (Ciccia et al., (D) ZRANB3/AH2 exhibits structure-specific endonuclease activity on the (2011). EMBO Rep. 12, 574–580. leading strand of replication fork structures in vitro (green triangle), which, in 2012, Yuan et al., 2012). conjunction with fork regression, may lead to repair of the lesion at the stalled Finally, although ZRANB3/ Ulrich, H.D., and Walden, H. (2010). replication fork. Nat. Rev. Mol. Cell Biol. 11, AH2 exhibits a specific pref479–489. erence for K63-polyubiquitinated PCNA, it is not clear whether ubiquitinate PCNA (Unk et al., 2010). Unk, I., Hajdu´, I., Blastya´k, A., and Haracska, L. ZRANB3/AH2 is functioning as a new What, then, is the role of ZRANB3/AH2? (2010). DNA Repair (Amst.) 9, 257–267. component in the DDT pathway, in a This may be clarified through DNA muta- Weston, R., Peeters, H., and Ahel, D. (2012). Genes parallel DNA repair pathway, or with tion analysis and epistasis studies with Dev. 15, 1558–1572. something else entirely. HLTF, a key ubiq- other DDT proteins. Electron microscopy Yuan, J., Ghosal, G., and Chen, J. (2012). Mol. Cell uitin ligase and translocase in the DDT and physical interaction studies may 47, this issue, 410–421. pathway, also has fork regression activity, also help reveal if ZRANB3/AH2 can Yusufzai, T., and Kadonaga, J.T. (2010). Proc. Natl. is important for fork restart, and can poly- actively promote fork regression in vivo, Acad. Sci. USA 107, 20970–20973.
334 Molecular Cell 47, August 10, 2012 ª2012 Elsevier Inc.