Investigating Mechanisms Of Genome Maintenance


To remain viable and healthy, an organism’s cells must carry out high fidelity DNA replication before every mitosis. Successful DNA replication faces two major challenges. One involves the vast amount of DNA to be copied, especially in higher eukaryotes. To meet this challenge, vertebrate cells initiate DNA replication from ~100,000 origins of replication in every S phase. A second challenge arises because DNA is continually damaged by exogenous and endogenous agents. As a result, replication forks often encounter DNA damage in the template strands. We seek to understand how cells faithfully duplicate their DNA in the presence and absence of DNA damage.

Single Molecule Imaging Cartoon
Single Molecule Imaging of DNA Replication

In eukaryotic cells, a complex of three factors (CDC45, MCM2-7, and GINS) forms the CMG helicase, which unwinds DNAat the replication fork. How CMG functions and how it responds to DNA damage remain unclear. To study DNA replication at the single molecule level, we immobilize λ DNA (50kB) in a microfluidic flow cell and inject frog egg extract, which promotes bi-directional DNA replication on a well-resolved DNA template [Yardimci et al. 2010 Mol Cell]Yardimci, H., Loveland, A.B., Habuchi, S., van Oijen, A.M., and Walter, J.C. (2010). Uncoupling of sister replisomes during eukaryotic DNA replication. Molecular cell 40, 834-840. Download .pdf. Using this approach, we showed that CMG pauses longer when confronted with a bulky adduct on the leading strand template versus the lagging strand template, arguing that CMG translocates along the leading strand [Fu et al. 2011 Cell]Fu, Y.V., Yardimci, H., Long, D.T., Guainazzi, A., Bermudez, V.P., Hurwitz, J., van Oijen, A., Scharer, O.D., and Walter, J.C. (2011). Selective Bypass of a Lagging Strand Roadblock by the Eukaryotic Replicative DNA Helicase. Cell 146, 931-941. Download .pdf. More recently, we have directly imaged fluorescently labeled CMG at the replication fork to monitor its dynamics in real time [Sparks et al. 2019 Cell]Sparks, J., Chistol, G., Gao., A.O., Raschle, M., Larsen., N.B., Mann, M., Duxin, J.P., and Walter, J.C. (2019). The CMG helicase bypasses DNA protein cross-links to facilitate their repair. Cell 176, 167–181. Download .pdf. We are using this approach to examine the fate of CMG after collision with different forms of DNA damage

ICL Repair cartoon
DNA Interstrand Cross-Link Repair

Our cells contain numerous metabolites that can generate DNA interstrand cross-links (ICLs), and chemotherapeutics such as cisplatinum are thought to kill cancer cells by inducing ICLs. ICL repair requires DNA replication and 22 “FANC” proteins, defects in which cause the bone marrow failure and cancer predisposition syndrome Fanconi anemia (FA). However, how the FANC proteins promote ICL repair was unknown. Using replication of a cispltain ICL-containing plasmid in egg extracts, we showed that ICL repair is triggered by the convergence of two replication forks on the lesion [Raschle et al. 2008 Cell]Raschle, M., Knipscheer, P., Enoiu, M., Angelov, T., Sun, J., Griffith, J.D., Ellenberger, T.E., Scharer, O.D., and Walter, J.C. (2008). Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134, 969-980. Download .pdf. After ubiquitylation of the CMG helicase, its extraction from chromatin by the p97 ATPase [Zhang et al. 2015 Nat Struct Mol Biol]Zhang, J., Dewar, J.M., Budzowska, M., Motnenko, A., Cohn, M.A., and Walter, J.C. (2015). DNA interstrand cross-link repair requires replication-fork convergence. Nat Struct Mol Biol 22, 242-247. Download .pdf, and the reversal of one fork [Amunugama et al. 2018 Cell Rep]Amunugama, R., Willcox, S., Wu, A., Abdullah, U.B., El-Sagheer, A.H., Brown, T., McHugh, P.J., Griffith, J.D., and Walter, J.C. (2018). Replication Fork Reversal During DNA Interstrand Crosslink Repair Requires CMG Unloading. Cell Rep 23, 3419-3428. Download .pdf, the cross-link is resolved via FANC protein-dependent dual incisions [Knipscheer et al. 2009 Science]Knipscheer, P., Raschle, M., Smogorzewska, A., Enoiu, M., Ho, T.V., Scharer, O.D., Elledge, S.J., and Walter, J.C. (2009). The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science 326, 1698-1701. Download .pdf [Klein Douwel et al. 2014 Mol Cell]Klein Douwel, D., Boonen, R.A., Long, D.T., Szypowska, A.A., Raschle, M., Walter, J.C., and Knipscheer, P. (2014). XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4. Mol Cell 54, 460-471. Download .pdf. These incisions create a double-strand break that is repaired via homologous recombination [Long et al. 2011 Science]Long, D.T., Raschle, M., Joukov, V., and Walter, J.C. (2011). Mechanism of RAD51-dependent DNA interstrand cross-link repair. Science 333, 84-87. Download .pdf. More recently, we discovered that other ICLs can be resolved by a fundamentally different pathway in which the DNA glycosylase NEIL3 resolves the ICL without formation of a DSB break intermediate [Semlow et al. 2016 Cell]Semlow, D.R., Zhang, J., Budzowska, M., Drohat, A.C., and Walter, J.C. (2016). Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase. Cell 167, 498-511. Download .pdf. Most recently, we discovered that the E3 ubiquitin ligase TRAIP ubiquitylates CMG at ICLs and thereby prioritizes the simple NEIL3 pathway over the more complicated Fanconi ICL repair pathway [Wu et al. 2019 Nature]Wu, R.A. , Semlow, D.R., Kamimae-Lanning, A.N., Kochenova, O.V., Chistol, G., Hodskinson, M.R., Amunugama, R., Sparks, J.L., Wang, M., Deng, L., Mimoso, C.A., Low, E., Patel, K.J., Walter, J.C. (2019). TRAIP is a master regulator of DNA interstrand crosslink repair. Nature. doi:10.1038/s41586-019-1002-0. Download .pdf.

DPC repair cartoon
DNA-Protein Cross-Link Repair

Most of the agents that generate ICLs also form DNA-protein cross-links (DPCs). Like ICLs, DPCs inhibit replication fork progression, but no dedicated DPC repair pathway was known. To establish a defined system for DPC repair, we covalently attached a 45 kD protein to a specific site on a plasmid and replicated it in egg extract. We showed that collision of a replication fork with this DPC led to DPC proteolysis, yielding a short DNA-peptide adduct that can be bypassed by the leading strand [Duxin et al. 2014 Cell]Duxin, J.P., Dewar, J.M., Yardimci, H., and Walter, J.C. (2014). Repair of a DNA-protein crosslink by replication-coupled proteolysis. Cell 159, 346-357. Download .pdf. Remarkably, assisted by RTEL1, the CMG helicase bypasses the intact DPC before the DPC undergoes proteolysis, providing an elegant mechanism to prevent CMG destruction, which would cause fork collapse [Sparks et al. 2019 Cell]Sparks, J., Chistol, G., Gao., A.O., Raschle, M., Larsen., N.B., Mann, M., Duxin, J.P., and Walter, J.C. (2019). The CMG helicase bypasses DNA protein cross-links to facilitate their repair. Cell 176, 167–181. Download .pdf. We are now using single-molecule imaging and ensemble assays to understand how the CMG helicase can bypass a massive adduct on its translocation strand.

Replication termination cartoon
Replication Termination

In vertebrate cells, replication terminates at ~100,000 sites in every S phase, yet the underlying mechanism was obscure. Studies of viral DNA replication suggested that converging replisomes clash, leading to a delay in the completion of DNA synthesis while replisomes are disassembled. Using a novel approach to induce site-specific termination in egg extracts, we described a new model of termination in which leading strands do not stall and replisome disassembly occurs after all DNA synthesis events have been completed [Dewar et al. 2015 Nature]Dewar, J.M., Budzowska, M., and Walter, J.C. (2015). The mechanism of DNA replication termination in vertebrates. Nature 525, 345-350. Download .pdf. We also discovered that the E3 ubiquitin ligase CRL2Lrr1 ubiquitylates the replisome and promotes its disassembly at the end of DNA replication [Dewar et al. 2017 Genes Dev]Dewar, J.M., Low, E., Mann, M., Raschle, M., and Walter, J.C. (2017). CRL2(Lrr1) promotes unloading of the vertebrate replisome from chromatin during replication termination. Genes Dev 31, 275-290. Download .pdf. We are now using single molecule imaging and other approaches to determine how CRL2Lrr1 specifically targets terminated CMGs while avoiding active CMGs, whose premature disassembly would cause fork collapse and genome instability. We are also investigating how the p97 ATPase disassembles the ubiquitylated replisome.

Non-Homologous End Joining Cartoon
Double-strand break repair

In collaboration with the laboratory of our neighbor Joe Loparo, we also work on the repair of DNA double-strand breaks (DSBs), which pose a severe threat to genome integrity. In eukaryotic cells, non-homologous end joining (NHEJ) repairs DSBs via direct ligation by XRCC4-LigaseIV but the underlying mechanism is incompletely understood. Using single molecule FRET in egg extracts, we showed that Ku and DNA-PKcs assemble DNA ends into a “long-range” synaptic complex in which DNA ends associate without causing FRET. Subsequently, XLF and XRCC4-LigaseIV promote the formation of a “short-range” complex in which fluorescently labeled DNA ends are closely aligned in a FRET-positive state [Graham et al. 2016 Mol Cell]Graham, T.G., Walter, J.C., and Loparo, J.J. (2016). Two-Stage Synapsis of DNA Ends during Non-homologous End Joining. Mol Cell 61, 850-858. Download .pdf. Often, DNA ends are chemically damaged and require enyzmatic processing before ligation can occur. We have shown that processing occurs primarily in the short-range complex. In this manner, ends undergo ligation as soon as they become chemically compatible, limiting mutations [Stinson et al., 2019 Mol. Cell]Stinson, B.M., Moreno, A.T., Walter, J.C., Loparo, J.J.A Mechanism to Minimize Errors during Non-homologous End Joining. . Download .pdf.