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Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Kang YH, Munashingha PR, Lee CH, Nguyen TA, Seo YS - Nucleic Acids Res. (2011)

Bottom Line: Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase.For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap.We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Center for DNA Replication and Genome Instability, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.

ABSTRACT
Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

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Model of Mph1 function on stalled replication forks arising from leading strand damage template and mode of Mph1 action in D-loop displacement. (A) A model describing the role of Mph1 in restarting stalled replication forks (see the text for detailed description). (B) Illustration of three representative D-loop substrates (5′-, 3′- or no-tailed) used previously (6). The arrows indicate the structure equivalent to flap structure that can be used for displacement of invaded strand (3′ or double flap as described).
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gkr983-F10: Model of Mph1 function on stalled replication forks arising from leading strand damage template and mode of Mph1 action in D-loop displacement. (A) A model describing the role of Mph1 in restarting stalled replication forks (see the text for detailed description). (B) Illustration of three representative D-loop substrates (5′-, 3′- or no-tailed) used previously (6). The arrows indicate the structure equivalent to flap structure that can be used for displacement of invaded strand (3′ or double flap as described).

Mentions: Mph1 was shown to play a role in damage avoidance when DNA replication forks are stalled due to damage lesions in the template as (5). A similar role for the fission yeast homolog Fml1 in this regard was recently proposed (9); it was suggested that stalled forks lead to the formation of a four-way junction by the regression of stalled replication forks when leading strand DNA synthesis is blocked by a damage on template DNA. Our analyses of Mph1 helicase activity suggest that the action of Mph1 leads to fork regression as illustrated in Figure 10A. (i) Mph1 first recognizes stalled replication forks with ssDNA regions exposed. (ii) Mph1 binds to the exposed ssDNA region and displaces the 5′-end of the nascent lagging strand. (iii) This then permits both template strands to re-anneal, resulting in the regression of replication forks for error-free repair of the lesion. This scenario is compatible with a proposed model based on studies with Fml1 in which DNA lesions were shown to be bypassed by using the nascent lagging strand as template (9). (iv) The final step in our model leads to the restoration of regressed replication forks. This could be achieved by Mph1 catalyzing the reversal of the regressed replication fork. We showed that Mph1 could branch-migrate a four-way junction substrate in vitro in the direction leading to the preferential removal of the shortest arm (Figure 7B and C). These are also discussed in recent publications (37–39).Figure 10.


Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Kang YH, Munashingha PR, Lee CH, Nguyen TA, Seo YS - Nucleic Acids Res. (2011)

Model of Mph1 function on stalled replication forks arising from leading strand damage template and mode of Mph1 action in D-loop displacement. (A) A model describing the role of Mph1 in restarting stalled replication forks (see the text for detailed description). (B) Illustration of three representative D-loop substrates (5′-, 3′- or no-tailed) used previously (6). The arrows indicate the structure equivalent to flap structure that can be used for displacement of invaded strand (3′ or double flap as described).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300029&req=5

gkr983-F10: Model of Mph1 function on stalled replication forks arising from leading strand damage template and mode of Mph1 action in D-loop displacement. (A) A model describing the role of Mph1 in restarting stalled replication forks (see the text for detailed description). (B) Illustration of three representative D-loop substrates (5′-, 3′- or no-tailed) used previously (6). The arrows indicate the structure equivalent to flap structure that can be used for displacement of invaded strand (3′ or double flap as described).
Mentions: Mph1 was shown to play a role in damage avoidance when DNA replication forks are stalled due to damage lesions in the template as (5). A similar role for the fission yeast homolog Fml1 in this regard was recently proposed (9); it was suggested that stalled forks lead to the formation of a four-way junction by the regression of stalled replication forks when leading strand DNA synthesis is blocked by a damage on template DNA. Our analyses of Mph1 helicase activity suggest that the action of Mph1 leads to fork regression as illustrated in Figure 10A. (i) Mph1 first recognizes stalled replication forks with ssDNA regions exposed. (ii) Mph1 binds to the exposed ssDNA region and displaces the 5′-end of the nascent lagging strand. (iii) This then permits both template strands to re-anneal, resulting in the regression of replication forks for error-free repair of the lesion. This scenario is compatible with a proposed model based on studies with Fml1 in which DNA lesions were shown to be bypassed by using the nascent lagging strand as template (9). (iv) The final step in our model leads to the restoration of regressed replication forks. This could be achieved by Mph1 catalyzing the reversal of the regressed replication fork. We showed that Mph1 could branch-migrate a four-way junction substrate in vitro in the direction leading to the preferential removal of the shortest arm (Figure 7B and C). These are also discussed in recent publications (37–39).Figure 10.

Bottom Line: Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase.For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap.We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Center for DNA Replication and Genome Instability, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.

ABSTRACT
Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

Show MeSH
Related in: MedlinePlus