RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the 5′ Terminus

The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1–2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000–2000 nucleotides downstream (5′ → 3′) of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5′-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.

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Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling

10.1101/2021.11.08.467732 ◽

2021 ◽

Author(s):

Tanay Thakar ◽

Joshua Straka ◽

Claudia M Nicolae ◽

George-Lucian Moldovan

Keyword(s):

Genome Instability ◽

Replication Stress ◽

Dependent Manner ◽

Replication Forks ◽

Nucleosome Assembly ◽

Critical Determinant ◽

Single Stranded Dna ◽

Histone Deposition ◽

Stalled Replication Forks ◽

Lagging Strand

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Polα-dependent lagging strand gaps, which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.

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Faculty Opinions recommendation of Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks.

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

10.3410/f.718358026.793494295 ◽

2014 ◽

Author(s):

Domenico Maiorano

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Replication Forks ◽

Single Stranded Dna ◽

The Stability ◽

Stalled Replication Forks

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Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Nucleic Acids Research ◽

10.1093/nar/gku276 ◽

2014 ◽

Vol 42 (10) ◽

pp. 6326-6336 ◽

Cited By ~ 31

Author(s):

Emma Bolderson ◽

Eva Petermann ◽

Laura Croft ◽

Amila Suraweera ◽

Raj K. Pandita ◽

...

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Replication Forks ◽

Single Stranded Dna ◽

The Stability ◽

Stalled Replication Forks

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On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)32965-x ◽

1983 ◽

Vol 258 (4) ◽

pp. 2577-2585 ◽

Cited By ~ 12

Author(s):

M M Cox ◽

D A Soltis ◽

Z Livneh ◽

I R Lehman

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Reca Protein ◽

Dna Binding Protein ◽

Strand Exchange ◽

Single Stranded Dna ◽

Dna Strand Exchange ◽

Dna Strand

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The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

Nature Communications ◽

10.1038/s41467-020-20818-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ann-Marie K. Shorrocks ◽

Samuel E. Jones ◽

Kaima Tsukada ◽

Carl A. Morrow ◽

Zoulikha Belblidia ◽

...

Keyword(s):

Replication Stress ◽

Bloom Syndrome ◽

Holliday Junctions ◽

Replication Forks ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Dna Replication Stress ◽

Dna End Resection ◽

Stalled Replication Forks

AbstractThe Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

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Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805307105 ◽

2008 ◽

Vol 105 (35) ◽

pp. 12973-12978 ◽

Cited By ~ 17

Author(s):

N. Nitani ◽

C. Yadani ◽

H. Yabuuchi ◽

H. Masukata ◽

T. Nakagawa

Keyword(s):

Replication Forks ◽

Single Stranded Dna ◽

Terminal Domain ◽

Mcm Helicase ◽

Stalled Replication Forks

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Structural basis of HMCES interactions with DNA reveals multivalent substrate recognition

10.1101/519595 ◽

2019 ◽

Author(s):

Levon Halabelian ◽

Mani Ravichandran ◽

Yanjun Li ◽

Hong Zheng ◽

L. Aravind ◽

...

Keyword(s):

Dna Damage ◽

Crystal Structures ◽

Genome Instability ◽

Substrate Recognition ◽

Catalytic Triad ◽

Structural Basis ◽

Replication Forks ◽

Abasic Sites ◽

Stalled Replication Forks ◽

Gapped Dna

ABSTRACTHMCES can covalently crosslink to abasic sites in single-stranded DNA at stalled replication forks to prevent genome instability. Here, we report crystal structures of the HMCES SRAP domain in complex with DNA-damage substrates, revealing interactions with both single-stranded and duplex segments of 3’ overhang DNA. HMCES may also bind gapped DNA and 5’ overhang structures to align single stranded abasic sites for crosslinking to the conserved Cys2 of its catalytic triad.

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Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.4.1350-1356.2005 ◽

2005 ◽

Vol 187 (4) ◽

pp. 1350-1356 ◽

Cited By ~ 28

Author(s):

Ivana Ivančić-Baće ◽

Erika Salaj-Šmic ◽

Krunoslav Brčić-Kostić

Keyword(s):

Escherichia Coli ◽

Double Mutant ◽

Reca Protein ◽

Recq Helicase ◽

Single Stranded Dna ◽

Double Stranded Dna ◽

Recf Pathway ◽

Recombination Pathway ◽

Recbcd Enzyme ◽

Dna Ends

ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.

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