Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

Mapping Intimacies ◽

10.1101/2021.12.10.472094 ◽

2021 ◽

Author(s):

Debolina Bandyopadhyay ◽

Padmaja P Mishra

Keyword(s):

Single Molecule ◽

Vital Role ◽

Holliday Junction ◽

Asymmetric Mode ◽

Molecular Surveillance ◽

Efficient Gene ◽

Leading Strand ◽

Single Molecule Analysis ◽

Functional Mechanisms ◽

Stalled Fork

AbstractHelicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.

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Beginning at the end: DNA replication within the telomere

The Journal of Cell Biology ◽

10.1083/jcb.201506078 ◽

2015 ◽

Vol 210 (2) ◽

pp. 177-179 ◽

Cited By ~ 3

Author(s):

Susan A. Gerbi

Keyword(s):

Dna Replication ◽

Mouse Chromosome ◽

Single Molecule ◽

Template Strand ◽

Cell Biol ◽

Chromosome Arm ◽

Leading Strand ◽

Wrn Helicase ◽

Single Molecule Analysis

Using single molecule analysis of replicated DNA (SMARD), Drosopoulos et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201410061) report that DNA replication initiates at measurable frequency within the telomere of mouse chromosome arm 14q. They demonstrate that resolution of G4 structures on the G-rich template strand of the telomere requires some overlapping functions of BLM and WRN helicase for leading strand synthesis.

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BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

The Journal of Cell Biology ◽

10.1083/jcb.201410061 ◽

2015 ◽

Vol 210 (2) ◽

pp. 191-208 ◽

Cited By ~ 63

Author(s):

William C. Drosopoulos ◽

Settapong T. Kosiyatrakul ◽

Carl L. Schildkraut

Keyword(s):

Single Molecule ◽

Werner Syndrome ◽

Telomeric Dna ◽

G4 Dna ◽

Genome Wide ◽

Blm Helicase ◽

Telomere Replication ◽

Leading Strand ◽

Single Molecule Analysis

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

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