Advances in understanding DNA processing and protection at stalled replication forks

Rickman, Kimberly A.; Smogorzewska, Agata

doi:10.1083/jcb.201809012

Cited by 123 publications

(133 citation statements)

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“…While the role of MRE11 in nascent strand degradation of BRCA2 deficient cells has been widely shown, there is conflicting data about resection mediated by DNA2 [25,26,50]. A role for DNA2 with WRN in replication fork restart has been described, and it has also been reported that DNA2 degrades nascent DNA at stalled forks in the setting of RECQ1, BOD1L, or Abro1 deficiency [24,[51][52][53]. Here we show in isogenic cell lines that BRCA2 function is required to also prevent DNA2 resection at HU-stalled forks.…”

Section: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

“…Functional analysis of BRCA2 has largely focused on canonical HR and the role of BRCA2 in ICL repair has been associated with the repair of DSBs generated by programmed incisions at the ICL. Outside of their role in HR and ICL repair, BRCA2 and RAD51 along with a number of other recently described proteins, function in replication fork protection [24]. In the absence of replication fork protection, newly synthesized DNA is degraded at hydroxyurea (HU) stalled replication forks and a number of nucleases including MRE11, CTIP, and EXO1 have been implicated in the process [24][25][26].…”

Section: Introductionmentioning

confidence: 99%

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Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

Rickman

Noonan

Lach

et al. 2019

Preprint

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DNA interstrand crosslinks (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, results in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of the programmed DNA double strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA binding domain (DBD) mutants discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for protection of DNA at an ICL. Cells carrying DBD BRCA2 mutations are sensitive to ICL inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease implicated in the resection of HU-stalled replication forks. Our results also indicate that unlike the processing at HU-stalled forks, function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.2 Introduction:

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Section: Brca2 Function At the Hu-stalled Replication Forkmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

Rickman

Noonan

Lach

et al. 2019

Preprint

Self Cite

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“…[38][39][40] The finding that REV7 is also required for the resistance caused by TIP60 depletion supports a mechanism involving activity at the double-strand break; 41 however, other potential roles for REV7 at the replication fork have not yet been thoroughly characterized. 42 Therefore, other function(s) of 53BP1/REV7 may potentially be involved in the rescue of PARPi-induced cytotoxicity downstream of TIP60 depletion in BRCA2-deficient cells.…”

Section: Discussionmentioning

confidence: 99%

Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

Clements

Hale

Tolman

et al. 2019

Preprint

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Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in the homologous recombination (HR) DNA repair pathway, such as those lacking BRCA2. In light of this, PARPi have been utilized in recent years to treat BRCA2-mutant tumors, with many patients deriving impressive clinical benefit. However, positive response to PARPi is not universal, even among patients with HRdeficient tumors. Here, we present the results of three genome-wide CRISPR knockout and activation screens which provide an unbiased look at genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we reveal that depletion of the histone acetyltransferase TIP60, a top hit from our screens, robustly reverses the PARPi sensitivity caused by BRCA2 deficiency. Mechanistically, we show that TIP60 depletion rewires double strand break repair in BRCA2-deficient cells by promoting 53BP1 binding to double strand breaks to suppress end resection. Our work provides a comprehensive set of putative biomarkers that serve to better understand and predict PARPi response, and identifies a novel pathway of PARPi resistance in BRCA2-deficient cells.

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“…These include alternate DNA structures, protein: DNA adducts, DNA covalent modifications introduced by endogenous or endogenous reactants, depleted nucleotide precursor pools, etc. Replication stress activates the ATR (ATM- and Rad3-related) kinase, with hundreds of substrates, including MCM proteins 16–18 , and stimulates the recruitment of numerous factors to stalled replication forks 19–22 . These function in a variety of pathways to relieve obstacles, reconstruct broken forks, and restart replication.…”

Section: Introductionmentioning

confidence: 99%

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Zhang

Bellani

James

et al. 2020

Preprint

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25Duplication of mammalian genomes requires replisomes to overcome numerous impediments 26 during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of 27 replication stress characterize mixed populations of challenged and unchallenged replication forks, 28 averaged across S phase, and model a single species of "stressed" replisome. However, in cells 29 containing potent obstacles to replication, we find two different lesion proximal replisomes. One 30 is bound by the DONSON protein and is more frequent in early S phase, in regions marked by 31 euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S 32 phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-33 seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions 34 that replicate early and the skew of the latter towards regions that replicate late. 35Introduction 36 Eukaryotic replisomes are multiprotein complexes consisting, minimally, of the CMG 37 helicase [MCM2-7 (M), CDC45 (C), and GINS (go, ichi, ni, san) proteins (G)] which forms a ring 38 around the leading strand template. Other components include the pol α, ε, and δ polymerases, 39 MCM10, and a few accessory factors [1][2][3][4][5][6][7] . The identification and characterization of the minimal 40 components of biochemically active replisomes, the result of decades of extraordinary work from 41 multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under 42 carefully controlled conditions. However, in vivo there are hundreds of replisome associated 43 proteins 8-12 . Presumably this reflects the multiple layers of complexity that characterize replication 44 of the genome in living cells. For example, three dimensional analyses of chromosome structure 45 demonstrate two major domains. The A compartment contains euchromatin, which is accessible, 46 transcriptionally active, and marked by specific histone modifications, such as H3K4me3. The B 47 compartment, which is more condensed, contains inactive genes, many repeated elements, and is 48 associated with different histone modifications, including H3K9me3 13 . In addition to the structural 49 distinctions, regions of the genome are also subject to temporal control of replication during S 50 phase. Sequences in Compartment A tend to replicate early in S phase, while those in B are 51 duplicated in late S phase 14,15 . 52 Other influential effectors of replisome composition are the frequent encounters with 53 impediments, that stall or block either the progress of the CMG helicase or DNA synthesis. These 54 3 include alternate DNA structures, protein: DNA adducts, DNA covalent modifications introduced 55 by endogenous or endogenous reactants, depleted nucleotide precursor pools, etc. Replication 56 stress activates the ATR (ATM-and Rad3-related) kinase, with hundreds of substrates, including 57 MCM proteins [16][17][18] , and stimulates the recruitment of numerous facto...

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Advances in understanding DNA processing and protection at stalled replication forks

Cited by 123 publications

References 104 publications

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

Identification of regulators of poly-ADP-ribose polymerase (PARP) inhibitor response through complementary CRISPR knockout and activation screens

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

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