The information necessary for the proper functioning of cells is stored in genomic DNA. Thus, it is crucial to maintain its integrity during cell division to faithfully transfer genetic information to daughter cells. However, genomic DNA is continuously subjected to spontaneous damage from reactive metabolites and environmental mutagens. Despite the protection provided by cellular DNA repair pathways, some damage may evade detection and persist into S-phase. However, replicative polymerases have very stringent polymerase domains as well as 3’ to 5’ exonuclease (“proofreading”) domains and thus cannot accommodate damaged bases. Consequently, upon encountering a damaged template base, progression of the replication fork is stalled or completely blocked, causing it to collapse. Failure to restart often results in double-strand breaks that may lead to gross chromosomal rearrangements, cell-cycle arrest, and cell death.
Therefore, it is often more advantageous to circumvent such replicative arrests and postpone repair of the offending damage to complete the cell cycle and maintain cell survival. Such a process, referred to as DNA damage tolerance (DDT) or lesion bypass, may be carried out by one of two pathways. In translesion DNA synthesis (TLS), the replicative polymerase is switched out for a specialized TLS polymerase that binds to the processivity sliding clamp, PCNA, and replicates past the damage. In template switching, it is suggested that enzyme-catalyzed regression of the replication fork creates a “chicken-foot” structure in which the original template strands are re-annealed and the stalled primer terminus utilizes the newly synthesized strand of the sister chromatid as a damage-free template. Subsequent reversal of the “chicken-foot” structure reconstitutes the normal replication fork at a point beyond the site of damage, effectively bypassing the block in an error-free manner. Both DDT pathways have been observed in organisms ranging from E. coli to humans. Furthermore, many of the components, the TLS polymerases in particular, are well-conserved.
Eukaryotic Translesion Synthesis
Characterized by a more “open” polymerase active site and the lack of an associated proofreading activity, TLS polymerases are able to support stable nucleotide incorporation opposite damaged templates, allowing replication to proceed. However, due to the varying levels of fidelity of TLS polymerases with the numerous forms of DNA damage, TLS is potentially mutagenic and is indeed responsible for many cellular point mutations. Thus, TLS must be tightly regulated to minimize the replicative input of TLS polymerases. Central to this regulation is the attachment of single ubiquitin moieties to PCNA, a process referred to as monoubiquination, and the so-called “polymerase switching.” We are interested in studying the underlying mechanism(s) of each process within the human system. In particular, we are interested in the coordination of the involved proteins and defining the dynamic interplay between these two processes in both time and space.
Monoubiquitination of PCNA by Rad6/Rad18
Lesion bypass synthesis is initiated by substituting a TLS polymerase for the high-fidelity replicative polymerase that is incapable of synthesis across damaged DNA. This exchange is believed to depend on the ubiquitination of PCNA in the stalled holoenzyme. The post-translational modification of PCNA involves monoubiquitination at residue K164, presumably on each of the three PCNA subunits catalyzed by the Rad6/Rad18 complex. Hence, we will investigate how stalling of the replicative holoenzyme by a damaged template is recognized by the ubiquitinating enzyme complexes. Does the buildup of RPA on ssDNA in front of the stalled holoenzyme affect the ubiquitination of the PCNA? Does Polη binding to Rad6/Rad18 influence PCNA ubiquitination? Mechanistic analyses of PCNA ubiquitination involved in polymerase exchange at a stalled replication site will be carried out both at the ensemble and single-molecule levels. A second related question concerns the role and regulation of Rad6/Rad18 in TLS. While Rad6/Rad18 is known to be necessary for the monoubiquitination of PCNA, other roles have been proposed such as the recruitment of Polη. The nature of the PCNA complex that is ubiquitinated is also unknown and can be probed using enzymatic and kinetic assays.
- Hedglin M., Benkovic S.J. “Regulation of Rad6/Rad18 Activity During DNA Damage Tolerance” (2015) Accepted for Publication, Annual Review of Biophysics.
During high-fidelity DNA replication, eukaryotic replicative DNA pols (δ and ε) are anchored to PCNA, the homotrimeric sliding clamp, via their PCNA-interacting protein (PIP) motifs. This essential interaction has been mapped to the “front face” of each PCNA monomer, orienting the polymerase towards the 3’ end of a Primer-Template junction. Most human TLS polymerases also contain PCNA-binding domains and those with PIP motifs bind to the same interfaces on PCNA as the replicative polymerases. However, compared to the replicative polymerases, the binding affinity of the PIP motifs within TLS polymerases for PCNA is significantly reduced due to variations in key residues in the consensus PIP sequence. Thus, in order for TLS to proceed efficiently across sites of damage, polymerase switching must occur in which a high-affinity replicative polymerase is replaced with an appropriate, low-affinity TLS polymerase. Upon nucleotide incorporation opposite the damaged template, the switch must then be reversed to limit the replicative input of TLS polymerases and resume high-fidelity DNA replication. It is thought that the relatively low-affinity of TLS polymerases for PCNA is responsible for their distributive behavior and promotes reversal of the polymerase switch. How then is the low-affinity of TLS polymerases for PCNA accommodated during polymerase switching? Is monoubiquitination of PCNA involved? Perhaps, one or more of an ever-expanding repertoire of accessory proteins purported to have roles in TLS? Utilizing the lagging strand human DNA polymerase, δ, and the most prominently studied human TLS polymerase, η, we wish to address these and other questions through various ensemble and single-molecule biochemical studies.
- Hedglin M., Kumar R., Benkovic S.J. “Replication Clamps and Clamp Loaders.” (2013) In M. DePamphilis, S. Bell, & M. Mechali (Eds), DNA Replication (pp. 165 – 183). Long Island, NY: Cold Spring Harbor Laboratory Press.
- Hedglin M., Perumal S.K., Hu Z., Benkovic S.J. “Stepwise Assembly of the Human Replicative Polymerase Holoenzyme.” (2013) eLife; 2:e00278*.
- Wang L, Xu X, Kumar R, Maiti B, Liu CT, Ivanov I, Lee TH, Benkovic SJ. “Probing DNA Clamps with Single-Molecule Force Spectroscopy.” (2013) Nucleic Acids Research, 41, 7804 – 7814.
- Kumar R, Nashine VC, Mishra PP, Benkovic SJ, Lee TH. “Stepwise Loading of Yeast Clamp Revealed by Ensemble and Single-molecule Studies.” (2010) PNAS, 107, 19736 – 19741.
Lesion bypass in T4 bacteriophage
While damages in the lagging strand are less likely to block fork progression and can be overcome through formation of a ssDNA gap that is repaired after fork passage; damage in the leading strand is a particular challenge due to the continuous nature of the leading-strand synthesis. In the T4 system, when the replisome encounters a lesion in the leading strand, replication in the lagging strand continues at least one Okazaki fragment beyond the lesion site. This uncoupled synthesis results in a DNA structure that can be effectively regressed by a DNA repair helicase to generate a Holliday junction structure required for template switching.
The T4 bacteriophage lacks translesion polymerases capable of carrying out lesion bypass synthesis and, unlike Escherichia coli, cannot reinitiate synthesis by re-priming the leading strand. Hence the T4 phage may exclusively use fork regression to bypass leading-strand lesions. Recent in vivo studies in T4 bacteriophage support the role of fork regression in stalled replication fork processing and reactivation via an active helicase driven pathway involving the UvsW protein.
This pathway involves the reactivation of stalled forks with leading-strand lesions employing remodeling of the replication fork at the lesion site. The details of such a pathway are poorly understood mainly owing to the difficulty of following simultaneously the activities of the different proteins involved in the process. Moreover, the presence of different overlapping repair mechanisms in vivo further complicates the evaluation of the relevance of fork regression pathways in DNA repair. In particular, the biological role of regressed forks in vivo, pathological structures versus physiological intermediates has been under vigorous debate.
We are interested in studying the lesion bypass mediated by fork regression and progression involved in stalled replication fork processing using a variety of ensemble and single-molecule kinetics and other biochemical and biophysical techniques.
We employ a variety of techniques including ensemble steady-state and pre-steady state (stopped-flow and rapid quench) analyses based on fluorescence resonance energy transfer (FRET), radiometric, fluorescence, and UV-visible absorbance detection, as well as, total internal reflection fluorescence (TIRF), fluorophore photobleaching, and magnetic tweezers based single-molecule measurements. We also use isothermal titration calorimetry (ITC), cross-linking, analytical ultracentrifugation (AUC), surface plasmon resonance (SPR) tounderstand these complex biochemical pathways involving protein-protein and protein-DNA interactions.
- Perumal, S.K., Nelson, S.W., Benkovic, S.J. (2013) Functional and Physical interaction of T4 UvsW helicase and Single-stranded DNA binding Protein gp32 through its C- terminal Acidic tail. J. Mol. Biol. 425, 2823-2839.
- Manosas, M., Perumal, S.K., Bianco, P., Retort, F., Benkovic, S.J., Croquette, V. (2013) RecG and UvsW catalyze efficient reannealing reactions critical for rescue of stalled replication forks. Nature Communications. 4:2368, 1-11, doi: 10.1038/ncomms3368.
- Manosas, M., Perumal, S.K., Croquette, V., Benkovic, S.J. (2012) Direct observation of stalled fork restart and lesion bypass via fork regression in the T4 replication system. Science 338, 1217-1220.
- Nelson S.W., Benkovic, S.J. (2010) Response of the bacteriophage T4 replisome to noncoding lesions and regression of a stalled replication fork. J. Mol. Biol. 401(5):743-56.
- Perumal, S.K., Raney, K.D., Benkovic, S.J. (2010) Analysis of the DNA translocation and unwinding activities of T4 phage helicases. Methods 51(3):277-88.
- Nelson S.W., Perumal, S.K., Benkovic, S.J. (2009) Processive and unidirectional translocation of monomeric UvsW helicase on single-stranded DNA. Biochemistry 48(5):1036-46.
- Nelson, S.W., Benkovic, S.J. (2007) The T4 phage UvsW protein contains both DNA unwinding and strand annealing activities. J. Biol. Chem. 282(1):407-16.
Dr. Mark Hedglin
Dr. Erin Noble
Dr. Binod Pandey
Dr. Senthil Perumal
Dr. Vincent Croquette, Laboratoire de Physique Statistique, Ecole Normale Supérieure, France