scholarly journals A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability

2021 ◽  
Author(s):  
Simona Graziano ◽  
Nuria Coll-Bonfill ◽  
Barbara Teodoro-Castro ◽  
Sahiti Kuppa ◽  
Jessica Jackson ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Binding Proteins ◽  
Physical Interaction ◽  
Ssdna Binding ◽  
Point Of Entry ◽  
Recombination Repair ◽  
Dna Strands ◽  
Increased Sensitivity ◽  
Ssdna Binding Proteins

Lamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.

scholarly journals Characterization of an SSB–dT25 complex: structural insights into the S-shaped ssDNA binding conformation

RSC Advances ◽  
2019 ◽  
Vol 9 (69) ◽  
pp. 40388-40396 ◽  
Author(s):  
Yen-Hua Huang ◽  
I-Chen Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Dna Replication ◽  
Binding Proteins ◽  
Ssdna Binding ◽  
Cellular Processes ◽  
Replication Restart ◽  
Recombination Repair ◽  
Complex Structural ◽  
Ssdna Binding Proteins ◽  
Structural Insights

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play an important role in all DNA-dependent cellular processes, such as DNA replication, recombination, repair, and replication restart.

scholarly journals Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

2006 ◽  
Vol 27 (5) ◽  
pp. 1592-1601 ◽  
Author(s):  
Naduparambil K. Jacob ◽  
Rachel Lescasse ◽  
Benjamin R. Linger ◽  
Carolyn M. Price
Keyword(s):  
Telomere Length ◽  
Binding Proteins ◽  
Growth Arrest ◽  
Cell Cycle Checkpoint ◽  
Cell Phenotype ◽  
Ssdna Binding ◽  
Cycle Checkpoint ◽  
Length Regulation ◽  
A Cell ◽  
Ssdna Binding Proteins

ABSTRACT The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3′ strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.

scholarly journals Assessing protein dynamics on low complexity single-strand DNA curtains

2018 ◽  
Author(s):  
Jeffrey M. Schaub ◽  
Hongshan Zhang ◽  
Michael M. Soniat ◽  
Ilya J. Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Binding Proteins ◽  
Low Complexity ◽  
Rolling Circle Replication ◽  
Sequence Composition ◽  
Ssdna Binding ◽  
Length Changes ◽  
Ssdna Binding Proteins

AbstractSingle-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. As ssDNA is more flexible than double-stranded (ds)DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription and repair.

scholarly journals Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

2018 ◽  
Author(s):  
Alexander Munden ◽  
Zhan Rong ◽  
Rama Gangula ◽  
Simon Mallal ◽  
Jared T. Nordman
Keyword(s):  
Copy Number ◽  
Replication Fork ◽  
Genome Stability ◽  
Replication Timing ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Replication Forks ◽  
Dna Copy Number ◽  
Replication Fork Progression ◽  
Maintain Genome Stability

ABSTRACTControl of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

scholarly journals A meiosis-specific factor C19orf57/4930432K21Rik/BRME1 modulates localization of RAD51 and DMC1 recombinases to DSBs in mouse meiotic recombination

2020 ◽  
Author(s):  
Kazumasa Takemoto ◽  
Naoki Tani ◽  
Yuki Takada-Horisawa ◽  
Sayoko Fujimura ◽  
Nobuhiro Tanno ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Meiotic Recombination ◽  
Binding Proteins ◽  
Strand Break ◽  
Specific Factor ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
Dna Double Strand Break ◽  
Multiple Processes ◽  
Ssdna Binding Proteins

SummaryMeiotic recombination is critical for genetic exchange and generation of chiasmata that ensures faithful chromosome segregation during meiosis I. Meiotic recombination is initiated by DNA double-strand break (DSB) followed by multiple processes of DNA repair. The exact mechanisms how recombinases localize to DSB remained elusive. Here we show that C19orf57/4930432K21Rik/BRME1 is a new player for meiotic recombination in mice. C19orf57/4930432K21Rik/BRME1 associates with ssDNA binding proteins, BRCA2 and MEILB2/HSF2BP, critical recruiters of recombinases onto DSB sites. Disruption of C19orf57/4930432K21Rik/BRME1 shows severe impact on DSB repair and male fertility. Remarkably, removal of single stranded DNA (ssDNA) binding proteins from DSB sites is delayed, and reciprocally the loading of RAD51 and DMC1 onto resected ssDNA is impaired in Brme1 KO spermatocytes. We propose that C19orf57/4930432K21Rik/BRME1 modulates localization of recombinases to meiotic DSB sites through the interaction with the BRCA2-MEILB2/HSF2BP complex during meiotic recombination.

Sign in / Sign up

Export Citation Format

Share Document