Vertebrate chromosomes are replicated from thousands of origins of replication. At each origin, two replication forks are established that travel in opposite directions, copying DNA as they go. When converging forks meet, replication terminates. Termination involves local completion of DNA synthesis, decatenation of daughter molecules, and replisome disassembly. Failure to properly execute these steps leads to genome instability, a hallmark of most cancers. Despite its importance, the mechanism of termination is poorly understood, largely because the timing and location of termination events is not well-defined. As a result, termination is difficult to study using conventional approaches such as chromatin immunoprecipitation. To overcome these challenges, we developed an approach to induce localized and synchronous termination events in Xenopus egg extracts. A plasmid containing an array of lac operator (lacO) sites is incubated with lac repressor (LacR) and added to egg extract. DNA replication initiates somewhere on the plasmid and two forks converge on the outer edges of the LacR array, where they stall. Upon addition of IPTG, LacR dissociates, forks immediately resume elongation, and they converge and terminate synchronously in a small region within the lacO array. Using this approach, we have undertaken the most detailed mechanistic dissection of termination to date, leading to a fundamentally new model of this process. We show that there is no detectable slowing of DNA synthesis as forks converge and that dissociation of the two replicative CMG helicases occurs after leading strands are ligated to downstream Okazaki fragments of the converging fork. This observation implies that when CMGs reach the downstream Okazaki fragment, they move from ssDNA onto dsDNA, from where they are unloaded. We also found that CMG unloading involves ubiquitylation of its MCM7 subunit and the action of the p97 ATPase. In this proposal, we will use our new cell-free system to further elucidate the mechanism of replication termination. We will test our novel hypothesis that the interaction of CMG with dsDNA triggers CMG unloading (Aim 1). We will identify the E3 ubiquitin ligase that ubiquitylates MCM7 to promote CMG unloading, as well as any adaptor proteins that cooperate with p97 to extract CMG from DNA (Aim 2). We will also determine how the E3 ubiquitin ligase and p97 recognize terminated CMGs. Finally, we will address how failure to unload CMG affects DNA replication and DNA repair (Aim 3). Our experiments will lead to the first comprehensive molecular description of eukaryotic replication termination and elucidate how the proper regulation of this process suppresses genome instability.

Public Health Relevance

To efficiently copy our vast genomes, cells start the process of DNA duplication at thousands of sites on the chromosome, and this process 'replication initiation' is beginning to be well understood. In this proposal, we will study how DNA replication finishes ('termination'), a process we know almost nothing about, but which is probably crucial to maintain genome integrity and prevent cancer.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM080676-09
Application #
9099351
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
Dewar, James M; Budzowska, Magda; Walter, Johannes C (2015) The mechanism of DNA replication termination in vertebrates. Nature 525:345-50
Joukov, Vladimir; Walter, Johannes C; De Nicolo, Arcangela (2014) The Cep192-organized aurora A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly. Mol Cell 55:578-91
Yardimci, Hasan; Walter, Johannes C (2014) Prereplication-complex formation: a molecular double take? Nat Struct Mol Biol 21:20-5
Slenn, Tamara J; Morris, Benjamin; Havens, Courtney G et al. (2014) Thymine DNA glycosylase is a CRL4Cdt2 substrate. J Biol Chem 289:23043-55
Long, David T; Joukov, Vladimir; Budzowska, Magda et al. (2014) BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork. Mol Cell 56:174-85
Havens, Courtney G; Shobnam, Nadia; Guarino, Estrella et al. (2012) Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2. J Biol Chem 287:11410-21
Enoiu, Milica; Ho, The Vinh; Long, David T et al. (2012) Construction of plasmids containing site-specific DNA interstrand cross-links for biochemical and cell biological studies. Methods Mol Biol 920:203-19
Salguero, Israel; Guarino, Estrella; Shepherd, Marianne E A et al. (2012) Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen. Curr Biol 22:720-6
Yardimci, Hasan; Loveland, Anna B; van Oijen, Antoine M et al. (2012) Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods 57:179-86
Raman, Malavika; Havens, Courtney G; Walter, Johannes C et al. (2011) A genome-wide screen identifies p97 as an essential regulator of DNA damage-dependent CDT1 destruction. Mol Cell 44:72-84

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