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Human RECQL5beta stimulates flap endonuclease 1.

Speina E, Dawut L, Hedayati M, Wang Z, May A, Schwendener S, Janscak P, Croteau DL, Bohr VA - Nucleic Acids Res. (2010)

Bottom Line: Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage.Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes.This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, National Institutes of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

ABSTRACT
Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

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RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates (A, Leading) or 5′ double-stranded flap substrate (B, Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). (C) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.
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Figure 6: RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates (A, Leading) or 5′ double-stranded flap substrate (B, Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). (C) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.

Mentions: The gap endonuclease (GEN) activity of FEN1 is critical for resolving stalled replication forks. It is responsible for cleavage of ssDNA regions that accumulate extensively in response to replication fork arrest (25). GEN activity specifically incises DNA replication-fork-like structures at the ssDNA region on either the lagging or the leading strand template. Therefore, DNA substrates were prepared with 3′ and 5′ double-stranded flap structures, which resemble DNA replication forks, and RECQL5β was tested for its ability to enhance FEN1 cleavage on such structures. FEN1 produced a GEN cleavage activity of 15% (2 nM FEN1) on the leading strand substrate (Figure 6A) and 24% (0.5 nM FEN1) on the lagging strand structure (Figure 6B, and C). There was also a strong 5′ exonuclease activity of FEN1 on the leading strand (Figure 6A). RECQL5β did not increase GEN cleavage on the leading strand in the concentration range of 0.5–8 nM and addition of ATP slightly decreased the rate of GEN cleavage (Figure 6A, lanes 3–7, and lanes 12–16). However, RECQL5β in the absence of ATP significantly increased GEN cleavage on the lagging strand, ∼2- to 2.7-fold (Figure 6B, lanes 5–7, and C). Addition of ATP resulted in less efficient stimulation of lagging strand GEN activity by RECQL5β (Figure 6B, lanes 12–16, and C). RECQL5β alone had no incision activity (Figure 6A and B, lanes 8 and 17).Figure 6.


Human RECQL5beta stimulates flap endonuclease 1.

Speina E, Dawut L, Hedayati M, Wang Z, May A, Schwendener S, Janscak P, Croteau DL, Bohr VA - Nucleic Acids Res. (2010)

RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates (A, Leading) or 5′ double-stranded flap substrate (B, Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). (C) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.
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Related In: Results  -  Collection

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Figure 6: RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates (A, Leading) or 5′ double-stranded flap substrate (B, Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). (C) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.
Mentions: The gap endonuclease (GEN) activity of FEN1 is critical for resolving stalled replication forks. It is responsible for cleavage of ssDNA regions that accumulate extensively in response to replication fork arrest (25). GEN activity specifically incises DNA replication-fork-like structures at the ssDNA region on either the lagging or the leading strand template. Therefore, DNA substrates were prepared with 3′ and 5′ double-stranded flap structures, which resemble DNA replication forks, and RECQL5β was tested for its ability to enhance FEN1 cleavage on such structures. FEN1 produced a GEN cleavage activity of 15% (2 nM FEN1) on the leading strand substrate (Figure 6A) and 24% (0.5 nM FEN1) on the lagging strand structure (Figure 6B, and C). There was also a strong 5′ exonuclease activity of FEN1 on the leading strand (Figure 6A). RECQL5β did not increase GEN cleavage on the leading strand in the concentration range of 0.5–8 nM and addition of ATP slightly decreased the rate of GEN cleavage (Figure 6A, lanes 3–7, and lanes 12–16). However, RECQL5β in the absence of ATP significantly increased GEN cleavage on the lagging strand, ∼2- to 2.7-fold (Figure 6B, lanes 5–7, and C). Addition of ATP resulted in less efficient stimulation of lagging strand GEN activity by RECQL5β (Figure 6B, lanes 12–16, and C). RECQL5β alone had no incision activity (Figure 6A and B, lanes 8 and 17).Figure 6.

Bottom Line: Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage.Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes.This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, National Institutes of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

ABSTRACT
Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

Show MeSH
Related in: MedlinePlus