scholarly journals Mycobacterium tuberculosisRecG Protein but Not RuvAB or RecA Protein Is Efficient at Remodeling the Stalled Replication Forks

2015 ◽  
Vol 290 (40) ◽  
pp. 24119-24139 ◽  
Author(s):  
Roshan Singh Thakur ◽  
Shivakumar Basavaraju ◽  
Jasbeer Singh Khanduja ◽  
K. Muniyappa ◽  
Ganesh Nagaraju
2012 ◽  
Vol 287 (42) ◽  
pp. 35621-35630 ◽  
Author(s):  
Katsumi Morimatsu ◽  
Yun Wu ◽  
Stephen C. Kowalczykowski

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.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoping Xu ◽  
Kai Ni ◽  
Yafeng He ◽  
Jianke Ren ◽  
Chongkui Sun ◽  
...  

AbstractThe Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.


2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.


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