Structural basis for the multi-activity factor Rad5 in replication stress tolerance

AbstractThe yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5’s activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5’s HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance.

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Structure of the HLTF HIRAN domain and its functional implications in regression of a stalled replication fork

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320008074 ◽

2020 ◽

Vol 76 (8) ◽

pp. 729-735

Author(s):

Asami Hishiki ◽

Mamoru Sato ◽

Hiroshi Hashimoto

Keyword(s):

Dna Binding ◽

Ubiquitin Ligase ◽

Replication Fork ◽

Structural Information ◽

E3 Ubiquitin Ligase ◽

Dna Helicase ◽

Structural Basis ◽

The Past ◽

Functional Implications ◽

Fork Regression

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog that is found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and is a pivotal protein in template-switched DNA synthesis that allows DNA replication to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. In addition, HLTF has a DNA-binding domain termed HIRAN (HIP116 and RAD5 N-terminal). HIRAN has been hypothesized to play a role in DNA binding; however, the structural basis of its role in DNA binding has remained unclear. In the past five years, several crystal structures of HIRAN have been reported. These structures revealed new insights into the molecular mechanism underlying DNA binding by HIRAN. Here, the structural information on HIRAN is summarized and the function of HIRAN in recognizing the 3′-terminus of the daughter strand at a stalled replication fork and the implications for its involvement in fork regression are discussed.

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Mms1 and Mms22 stabilize the replisome during replication stress

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0848 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2396-2408 ◽

Cited By ~ 13

Author(s):

Jessica A. Vaisica ◽

Anastasija Baryshnikova ◽

Michael Costanzo ◽

Charles Boone ◽

Grant W. Brown

Keyword(s):

Dna Replication ◽

Ubiquitin Ligase ◽

Replication Fork ◽

E3 Ubiquitin Ligase ◽

Replication Stress ◽

Replication Forks ◽

Replication Fork Progression ◽

Binding Partners ◽

Efficient Recovery ◽

Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

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MUS81 nuclease activity is essential for replication stress tolerance and chromosome segregation in BRCA2-deficient cells

Nature Communications ◽

10.1038/ncomms15983 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 39

Author(s):

Xianning Lai ◽

Ronan Broderick ◽

Valérie Bergoglio ◽

Jutta Zimmer ◽

Sophie Badie ◽

...

Keyword(s):

Stress Tolerance ◽

Chromosome Segregation ◽

Replication Fork ◽

Replication Stress ◽

Nuclease Activity ◽

Dna Lesions ◽

Genome Replication ◽

Replication Fork Progression ◽

Nucleolytic Activity ◽

Genomic Regions

AbstractFailure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

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Non-enzymatic roles of human RAD51 at stalled replication forks

Nature Communications ◽

10.1038/s41467-019-12297-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

Jennifer M. Mason ◽

Yuen-Ling Chan ◽

Ralph W. Weichselbaum ◽

Douglas K. Bishop

Keyword(s):

Replication Fork ◽

Replication Stress ◽

Strand Exchange ◽

Replication Forks ◽

Enzymatic Function ◽

Replication Restart ◽

D Loop ◽

Fork Regression ◽

Stalled Replication Forks ◽

Exchange Activity

Abstract The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork regression, supporting a model in which fork regression depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

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Structure of HIRAN domain of human HLTF bound to duplex DNA provides structural basis for DNA unwinding to initiate replication fork regression

The Journal of Biochemistry ◽

10.1093/jb/mvaa008 ◽

2020 ◽

Vol 167 (6) ◽

pp. 597-602

Author(s):

Asami Hishiki ◽

Mamoru Sato ◽

Hiroshi Hashimoto

Keyword(s):

Hydroxy Group ◽

Replication Fork ◽

Dna Lesions ◽

Structural Basis ◽

Duplex Dna ◽

Double Stranded Dna ◽

Strand Separation ◽

Fork Regression ◽

Stalled Fork ◽

Dsdna Binding

Abstract Replication fork regression is a mechanism to rescue a stalled fork by various replication stresses, such as DNA lesions. Helicase-like transcription factor, a SNF2 translocase, plays a central role in the fork regression and its N-terminal domain, HIRAN (HIP116 and Rad5 N-terminal), binds the 3’-hydroxy group of single-stranded DNA. Furthermore, HIRAN is supposed to bind double-stranded DNA (dsDNA) and involved in strand separation in the fork regression, whereas structural basis for mechanisms underlying dsDNA binding and strand separation by HIRAN are still unclear. Here, we report the crystal structure of HIRAN bound to duplex DNA. The structure reveals that HIRAN binds the 3’-hydroxy group of DNA and unexpectedly unwinds three nucleobases of the duplex. Phe-142 is involved in the dsDNA binding and the strand separation. In addition, the structure unravels the mechanism underlying sequence-independent recognition for purine bases by HIRAN, where the N-glycosidic bond adopts syn conformation. Our findings indicate direct involvement of HIRAN in the fork regression by separating of the daughter strand from the parental template.

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