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

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

Nature Communications ◽

10.1038/s41467-020-20538-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Miaomiao Shen ◽

Nalini Dhingra ◽

Quan Wang ◽

Chen Cheng ◽

Songbiao Zhu ◽

...

Keyword(s):

Stress Tolerance ◽

Ubiquitin Ligase ◽

Replication Fork ◽

Replication Stress ◽

Spatial Arrangement ◽

Structural Basis ◽

Dna Lesion ◽

Activity Factor ◽

Fork Regression ◽

New Type

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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Role of Double-Stranded DNA Translocase Activity of Human HLTF in Replication of Damaged DNA

Molecular and Cellular Biology ◽

10.1128/mcb.00863-09 ◽

2009 ◽

Vol 30 (3) ◽

pp. 684-693 ◽

Cited By ~ 115

Author(s):

András Blastyák ◽

Ildikó Hajdú ◽

Ildikó Unk ◽

Lajos Haracska

Keyword(s):

Translesion Synthesis ◽

Dna Lesions ◽

Template Switching ◽

Replication Forks ◽

Double Stranded Dna ◽

Fork Regression ◽

Dna Translocase ◽

Damaged Dna

ABSTRACT Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

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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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Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

10.1101/2019.12.18.880690 ◽

2019 ◽

Cited By ~ 1

Author(s):

Domagoj Baretić ◽

Michael Jenkyn-Bedford ◽

Valentina Aria ◽

Giuseppe Cannone ◽

Mark Skehel ◽

...

Keyword(s):

High Resolution ◽

Replication Fork ◽

Genome Stability ◽

Chromosome Replication ◽

Duplex Dna ◽

Multiple Factors ◽

Strand Separation ◽

Fork Protection Complex ◽

Efficient Replication ◽

Maintain Genome Stability

AbstractThe eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), that have important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3-3.5 Å resolution cryo-EM structures comprising CMG, Ctf4, Csm3/Tof1 and Mrc1 at a replication fork. The structures provide high-resolution views of CMG:DNA interactions, revealing the mechanism of strand separation. Furthermore, they illustrate the topology of Mrc1 in the replisome and show Csm3/Tof1 ‘grips’ duplex DNA ahead of CMG via a network of interactions that are important for efficient replication fork pausing. Our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

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Faculty Opinions recommendation of DNA damage-induced replication fork regression and processing in Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011878.186471 ◽

2003 ◽

Author(s):

Martin Marinus

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Replication Fork ◽

Fork Regression

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Faculty Opinions recommendation of Structural basis for DNA strand separation by a hexameric replicative helicase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725696173.793513515 ◽

2016 ◽

Author(s):

Michael Trakselis

Keyword(s):

Structural Basis ◽

Replicative Helicase ◽

Strand Separation ◽

Dna Strand

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RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽

10.1093/genetics/166.4.1631 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1631-1640 ◽

Cited By ~ 4

Author(s):

Janet R Donaldson ◽

Charmain T Courcelle ◽

Justin Courcelle

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Dna Lesions ◽

Replication Forks ◽

Wild Type ◽

Replication Machinery ◽

Dna Junctions ◽

Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

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Structural basis of TFIIH activation for nucleotide excision repair

10.1101/628032 ◽

2019 ◽

Author(s):

Goran Kokic ◽

Aleksandar Chernev ◽

Dimitry Tegunov ◽

Christian Dienemann ◽

Henning Urlaub ◽

...

Keyword(s):

Dna Repair ◽

Transcription Factor ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Lesions ◽

Structural Basis ◽

Disease Mutations ◽

Mechanistic Analysis ◽

Nucleotide Excision ◽

Dna Complex

AbstractGenomes are constantly threatened by DNA damage, but cells can remove a large variety of DNA lesions by nucleotide excision repair (NER)1. Mutations in NER factors compromise cellular fitness and cause human diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy2,3. The NER machinery is built around the multisubunit transcription factor IIH (TFIIH), which opens the DNA repair bubble, scans for the lesion, and coordinates excision of the damaged DNA single strand fragment1,4. TFIIH consists of a kinase module and a core module that contains the ATPases XPB and XPD5. Here we prepare recombinant human TFIIH and show that XPB and XPD are stimulated by the additional NER factors XPA and XPG, respectively. We then determine the cryo-electron microscopy structure of the human core TFIIH-XPA-DNA complex at 3.6 Å resolution. The structure represents the lesion-scanning intermediate on the NER pathway and rationalizes the distinct phenotypes of disease mutations. It reveals that XPB and XPD bind double- and single-stranded DNA, respectively, consistent with their translocase and helicase activities. XPA forms a bridge between XPB and XPD, and retains the DNA at the 5’-edge of the repair bubble. Biochemical data and comparisons with prior structures6,7 explain how XPA and XPG can switch TFIIH from a transcription factor to a DNA repair factor. During transcription, the kinase module inhibits the repair helicase XPD8. For DNA repair, XPA dramatically rearranges the core TFIIH structure, which reorients the ATPases, releases the kinase module and displaces a ‘plug’ element from the DNA-binding pore in XPD. This enables XPD to move by ~80 Å, engage with DNA, and scan for the lesion in a XPG-stimulated manner. Our results provide the basis for a detailed mechanistic analysis of the NER mechanism.

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