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Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures.

Tafel AA, Wu L, McHugh PJ - J. Biol. Chem. (2011)

Bottom Line: Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures.When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A.Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

ABSTRACT
HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

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HEL308 preferentially unwinds model fork substrates with a ssDNA junction. A, top row, the 3′ overhang partial duplex, splayed arms, and model fork with nascent lagging strand substrates listed in supplemental Table 1 were 5′ end-labeled on the upper strand (5′ with asterisk), mock-treated (0 pmol/μl lane), or treated with increasing concentrations from 0.016 to 1 pmol/μl of HEL308 (0.016, 0.06, 0.25, and 1.00 pmol/μl). Bottom row, substrates were treated with a fixed concentration (0.5 pmol/μl) of HEL308 for the stated times. Lanes marked nt contain control substrate not treated with enzyme, and those marked B include boiled fully denatured substrate. The DNA substrates analyzed are schematically depicted at the bottom of the corresponding gels. The position of substrate (s or substr.) and generated products (p or prod.) are marked next to the gels. B and C, quantification of the data obtained in A expressed in terms of unwinding as a function of HEL308 concentration in B and as a function of incubation time using the fixed concentration of HEL308 in C. The experiments were repeated in triplicate, and the error bars show mean ± S.D.
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Figure 3: HEL308 preferentially unwinds model fork substrates with a ssDNA junction. A, top row, the 3′ overhang partial duplex, splayed arms, and model fork with nascent lagging strand substrates listed in supplemental Table 1 were 5′ end-labeled on the upper strand (5′ with asterisk), mock-treated (0 pmol/μl lane), or treated with increasing concentrations from 0.016 to 1 pmol/μl of HEL308 (0.016, 0.06, 0.25, and 1.00 pmol/μl). Bottom row, substrates were treated with a fixed concentration (0.5 pmol/μl) of HEL308 for the stated times. Lanes marked nt contain control substrate not treated with enzyme, and those marked B include boiled fully denatured substrate. The DNA substrates analyzed are schematically depicted at the bottom of the corresponding gels. The position of substrate (s or substr.) and generated products (p or prod.) are marked next to the gels. B and C, quantification of the data obtained in A expressed in terms of unwinding as a function of HEL308 concentration in B and as a function of incubation time using the fixed concentration of HEL308 in C. The experiments were repeated in triplicate, and the error bars show mean ± S.D.

Mentions: The ability to displace nascent strands from replication fork-like substrates and to bind HJ-like structures has been shown for the HEL308-like protein StoHjm from Sulfolobus tokodaii (30). Additionally, structure-specific annealing and the ability to regress model replication forks has been shown for this protein (31). Related biochemical activities, notably unwinding of the lagging strand on model fork substrates, have been reported for Hel308a from Methanothermobacter thermautotrophicus and Hjm from Pyrococcus furiosus (31–33). These findings prompted us to investigate whether HEL308 could process a variety of model structures that arise during replication and HR repair (Fig. 2, B and C). HEL308 was unable to process either model four-way HJ or chicken foot-like structures but could efficiently unwind a splayed arms structure (Fig. 2B and data not shown). Indeed, the splayed arms structure was unwound more efficiently than the partial duplex with a 3′ overhang (Fig. 3) suggesting that HEL308 prefers branched structures akin to those found at replication forks. To investigate this further, we analyzed if HEL308 could process model fork structures that contain either a nascent leading and/or lagging strand (Figs. 2B and 3). Interestingly, the presence of a lagging strand stimulated the ability of HEL308 to unwind the splayed arm (Figs. 2B and 3). This effect was specific to the nascent lagging strand because the presence of a nascent leading strand had little effect on the ability of HEL308 to unwind the splayed arms (Fig. 2B), whereas the presence of both leading and lagging nascent strands inhibited HEL308 activity (C). The ability of HEL308 to preferentially unwind forks with a nascent lagging strand suggests that HEL308 might act at damaged replication forks in which DNA replication on the leading strand template has been stalled, resulting in polymerase uncoupling and the continued DNA synthesis on the lagging strand template.


Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures.

Tafel AA, Wu L, McHugh PJ - J. Biol. Chem. (2011)

HEL308 preferentially unwinds model fork substrates with a ssDNA junction. A, top row, the 3′ overhang partial duplex, splayed arms, and model fork with nascent lagging strand substrates listed in supplemental Table 1 were 5′ end-labeled on the upper strand (5′ with asterisk), mock-treated (0 pmol/μl lane), or treated with increasing concentrations from 0.016 to 1 pmol/μl of HEL308 (0.016, 0.06, 0.25, and 1.00 pmol/μl). Bottom row, substrates were treated with a fixed concentration (0.5 pmol/μl) of HEL308 for the stated times. Lanes marked nt contain control substrate not treated with enzyme, and those marked B include boiled fully denatured substrate. The DNA substrates analyzed are schematically depicted at the bottom of the corresponding gels. The position of substrate (s or substr.) and generated products (p or prod.) are marked next to the gels. B and C, quantification of the data obtained in A expressed in terms of unwinding as a function of HEL308 concentration in B and as a function of incubation time using the fixed concentration of HEL308 in C. The experiments were repeated in triplicate, and the error bars show mean ± S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091193&req=5

Figure 3: HEL308 preferentially unwinds model fork substrates with a ssDNA junction. A, top row, the 3′ overhang partial duplex, splayed arms, and model fork with nascent lagging strand substrates listed in supplemental Table 1 were 5′ end-labeled on the upper strand (5′ with asterisk), mock-treated (0 pmol/μl lane), or treated with increasing concentrations from 0.016 to 1 pmol/μl of HEL308 (0.016, 0.06, 0.25, and 1.00 pmol/μl). Bottom row, substrates were treated with a fixed concentration (0.5 pmol/μl) of HEL308 for the stated times. Lanes marked nt contain control substrate not treated with enzyme, and those marked B include boiled fully denatured substrate. The DNA substrates analyzed are schematically depicted at the bottom of the corresponding gels. The position of substrate (s or substr.) and generated products (p or prod.) are marked next to the gels. B and C, quantification of the data obtained in A expressed in terms of unwinding as a function of HEL308 concentration in B and as a function of incubation time using the fixed concentration of HEL308 in C. The experiments were repeated in triplicate, and the error bars show mean ± S.D.
Mentions: The ability to displace nascent strands from replication fork-like substrates and to bind HJ-like structures has been shown for the HEL308-like protein StoHjm from Sulfolobus tokodaii (30). Additionally, structure-specific annealing and the ability to regress model replication forks has been shown for this protein (31). Related biochemical activities, notably unwinding of the lagging strand on model fork substrates, have been reported for Hel308a from Methanothermobacter thermautotrophicus and Hjm from Pyrococcus furiosus (31–33). These findings prompted us to investigate whether HEL308 could process a variety of model structures that arise during replication and HR repair (Fig. 2, B and C). HEL308 was unable to process either model four-way HJ or chicken foot-like structures but could efficiently unwind a splayed arms structure (Fig. 2B and data not shown). Indeed, the splayed arms structure was unwound more efficiently than the partial duplex with a 3′ overhang (Fig. 3) suggesting that HEL308 prefers branched structures akin to those found at replication forks. To investigate this further, we analyzed if HEL308 could process model fork structures that contain either a nascent leading and/or lagging strand (Figs. 2B and 3). Interestingly, the presence of a lagging strand stimulated the ability of HEL308 to unwind the splayed arm (Figs. 2B and 3). This effect was specific to the nascent lagging strand because the presence of a nascent leading strand had little effect on the ability of HEL308 to unwind the splayed arms (Fig. 2B), whereas the presence of both leading and lagging nascent strands inhibited HEL308 activity (C). The ability of HEL308 to preferentially unwind forks with a nascent lagging strand suggests that HEL308 might act at damaged replication forks in which DNA replication on the leading strand template has been stalled, resulting in polymerase uncoupling and the continued DNA synthesis on the lagging strand template.

Bottom Line: Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures.When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A.Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

ABSTRACT
HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

Show MeSH
Related in: MedlinePlus