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Replication Protein A Blocks the Way Eric C. Greene* Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York 10032, United States
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replication apparatus might assemble at inappropriate locations, which could prevent proper access to the primer 3′ terminus. This could be a particular problem during translesion synthesis. Thus, the ability of PCNA to rapidly diffuse along DNA raises the question of what might keep this sliding clamp properly oriented at the primer/template junction long enough for it to engage the necessary replication machinery. To address this question, Hedglin and Benkovic developed an assay based upon fluorescence resonance energy transfer (FRET) to monitor the behavior of fluorescently tagged PNCA complexes loaded onto an oligonucleotide primer/template substrate using the clamp loader complex RFC (replication factor C).5 These substrates harbored a region of duplex DNA adjacent to a region of single-stranded DNA, reflecting a minimalist version of a replication intermediate. Using these FRET assays, the authors then confirmed that RFC could load PNCA onto the primer/template junction substrates and that PNCA then quickly dissociated from the free ends of these linear DNA substrates. Interestingly, when the authors blocked the double-stranded DNA end with a bulky biotin−streptavidin adduct, PCNA could still slide off of the single-stranded DNA end. However, naked single-stranded DNA is unlikely to exist in living cells. Instead, any single-stranded DNA that becomes transiently exposed, during any reaction related to normal eukaryotic DNA metabolism, will become quickly bound by a protein complex called RPA (replication protein A). RPA is an essential and highly abundant trimeric protein complex that has extremely high affinities for single-stranded DNA, and RPA is required for all biological processes involving a single-stranded DNA intermediate.6 Hedglin and Benkovic reasoned that in a normal physiological scenario, the single-stranded DNA present at the end of their substrate would be coated with RPA and that the presence of RPA itself might act as a steric block to prevent PCNA from sliding off of the single-stranded DNA end. Through a series of clever steady state and pre-steady state FRET assays, the authors convincingly demonstrated that RPA does in fact prevent PCNA from sliding off the single-stranded DNA end. These results support a new model in which RPA may play a regulatory role during DNA replication by ensuring that PNCA remains right where it is needed and preventing it from diffusing away from the primer/template junction. Similar findings have also been described for the Escherichia coli βclamp protein together with E. coli single-stranded binding protein (SSB),7 suggesting that the role of single-stranded DNA binding proteins in keeping sliding clamps in place is broadly conserved.
rocessive replication of genomic DNA requires the participation of sliding clamps, which act as processivity factors for replicative DNA polymerases by preventing them from prematurely falling off of the DNA. PCNA (proliferating cell nuclear antigen) serves as the eukaryotic sliding clamp, and this highly conserved protein forms a stable homotrimeric structure that completely encircles DNA.1 Similar ringlike architectures are conserved among replication sliding clamps from all domains of life. In addition to its role in promoting the processivity of replicative polymerases, PCNA is also necessary for the function of many specialized translesion DNA polymerases, which are required to allow replication through some types of DNA damage.2 This is especially true of bulky chemical adducts, which can block the progression of replicative polymerases and require the use of translesion polymerases. The mechanisms involved in the switch between replicative and translesion polymerases remain poorly understood. One possibility is that the replication machinery must stop and wait for the translesion polymerase to catalyze incorporation of a nucleotide at the bulk adducts, before normal replication can resume. Alternatively, the replicative polymerase might bypass the bulky adduct, leaving behind a gap that must then be filled by the translesion polymerase. For either model, the time required for completion of the underlying molecular events remains uncertain, but one can readily envision that in either case PCNA may have to wait at the damaged site for a relatively long period of time to allow for recognition and loading of the appropriate translesion polymerase. Given the remarkable structure of replication sliding clamps such as PCNA, it is easy to envision how their topological binding mechanism would confer a high degree of processivity to a DNA polymerase. However, to act as a processivity factor, it is essential that PCNA itself not bind tightly to the DNA through a traditional binding mechanism; otherwise, it might slow any polymerase to which it is bound. Instead, to effectively fulfill this role, PCNA must move freely along the DNA and provide little or no resistance as the replication machinery travels along the genome during DNA replication. Indeed, it is now known that PCNA and related sliding clamps can in fact diffuse very rapidly along naked DNA. For instance, bulk biochemical measurements have shown that PCNA, and other related sliding clamps, will quickly slide off of a DNA molecule if there is a free DNA end available for dissociation.3 The ability of sliding clamps to move along DNA has more recently been corroborated by single-molecule studies, which have revealed that PCNA can in fact move rapidly along DNA via onedimensional diffusion.4 Although central to the functional requirements of all sliding clamp proteins, the ability of PNCA to diffuse so quickly along DNA also raises a potential problem. Namely, if PCNA were to move away from its loading site too quickly, then the © 2017 American Chemical Society
Received: March 10, 2017 Published: March 23, 2017 1809
DOI: 10.1021/acs.biochem.7b00221 Biochemistry 2017, 56, 1809−1810
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Biochemistry This work represents an elegant example of using reductionist biochemistry to get at the inner workings of a complex biological apparatus. An interesting follow-up question will be to ask what prevents PCNA from sliding backward along the double-stranded potion of the DNA, Nucleosomes seem like a likely culprit, but this issue is perhaps worthy of experimental corroboration. More importantly, in the future, it will be essential to build upon these results by asking increasingly complex questions related to the mechanisms of eukaryotic replication and translesion synthesis. For instance, one can envision that similar FRET measurements may help define the timing of events such as polymerase recruitment or polymerase exchange during translesion synthesis. In addition, RPA must get out of the way as the polymerases bind to PCNA and begin synthesis. The authors’ experimental system may provide access to the structural rearrangements that must be taking place upon binding of the appropriate translesion polymerase and initial movement of this polymerase along the DNA template.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The author declares no competing financial interest.
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REFERENCES
(1) Bloom, L. B. (2009) Loading clamps for DNA replication and repair. DNA Repair 8, 570−578. (2) Goodman, M. F., and Woodgate, R. (2013) Translesion DNA polymerases. Cold Spring Harbor Perspect. Biol. 5, a010363. (3) Tinker, R. L., Kassavetis, G. A., and Geiduschek, E. P. (1994) Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA. EMBO J. 13, 5330−5337. (4) Kochaniak, A. B., Habuchi, S., Loparo, J. J., Chang, D. J., Cimprich, K. A., Walter, J. C., and van Oijen, A. M. (2009) Proliferating cell nuclear antigen uses two distinct modes to move along DNA. J. Biol. Chem. 284, 17700−17710. (5) Hedglin, M., and Benkovic, S. J. (2017) Replication protein A prohibits diffusion of the PCNA sliding clamp along single-stranded DNA. Biochemistry, DOI: 10.1021/acs.biochem.6b01213. (6) Chen, R., and Wold, M. S. (2014) Replication protein A: singlestranded DNA’s first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair. BioEssays 36, 1156−1161. (7) Laurence, T. A., Kwon, Y., Johnson, A., Hollars, C. W., O’Donnell, M., Camarero, J. A., and Barsky, D. (2008) Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy. J. Biol. Chem. 283, 22895−22906.
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DOI: 10.1021/acs.biochem.7b00221 Biochemistry 2017, 56, 1809−1810
