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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 stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). (A) A phosphorimage of a typical gel. (B) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.
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Figure 2: RECQL5β stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). (A) A phosphorimage of a typical gel. (B) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.

Mentions: RECQL5β was tested for its ability to stimulate FEN1 because WRN, BLM and RECQL4 have been shown to potently stimulate FEN1 (40–42, 46). The DNA substrate used in these studies consisted of a 19 bp duplex with a single unannealed 5′ nucleotide adjacent to an upstream 25 bp duplex (1 nt 5′-flap). The 1 nt 5′-flap substrate was susceptible to FEN1 cleavage and 2 nt and 1 nt products were produced (Figure 2A, lane 2), as previously reported (47). These products result from FEN1 cleavage at the junction between the flap and the downstream double-stranded DNA (dsDNA) and endonucleolytic cleavage of the first nucleotide in the downstream duplex DNA. In the presence of 5 nM FEN1, 12% of 1 nM substrate was incised (Figure 2A, lane 2, and B). Under these conditions, we analyzed FEN1 cleavage as a function of RECQL5β concentration. FEN1 cleavage was stimulated up to 7-fold (Figure 2A, lanes 3–7 and B). As expected, RECQL5β alone did not incise the substrate (Figure 2A, lane 8). To confirm that the presence of RECQL5β caused true stimulation rather than this being an effect of RECQL5β stabilization of FEN1 in the reaction, we did a control reaction with BSA and found no stimulation of FEN1. In the presence of 5 nM FEN1 and 20 nM BSA only 8% of 1 nM substrate was incised (Figure 2A, lane 9).Figure 2.


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 stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). (A) A phosphorimage of a typical gel. (B) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875029&req=5

Figure 2: RECQL5β stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). (A) A phosphorimage of a typical gel. (B) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.
Mentions: RECQL5β was tested for its ability to stimulate FEN1 because WRN, BLM and RECQL4 have been shown to potently stimulate FEN1 (40–42, 46). The DNA substrate used in these studies consisted of a 19 bp duplex with a single unannealed 5′ nucleotide adjacent to an upstream 25 bp duplex (1 nt 5′-flap). The 1 nt 5′-flap substrate was susceptible to FEN1 cleavage and 2 nt and 1 nt products were produced (Figure 2A, lane 2), as previously reported (47). These products result from FEN1 cleavage at the junction between the flap and the downstream double-stranded DNA (dsDNA) and endonucleolytic cleavage of the first nucleotide in the downstream duplex DNA. In the presence of 5 nM FEN1, 12% of 1 nM substrate was incised (Figure 2A, lane 2, and B). Under these conditions, we analyzed FEN1 cleavage as a function of RECQL5β concentration. FEN1 cleavage was stimulated up to 7-fold (Figure 2A, lanes 3–7 and B). As expected, RECQL5β alone did not incise the substrate (Figure 2A, lane 8). To confirm that the presence of RECQL5β caused true stimulation rather than this being an effect of RECQL5β stabilization of FEN1 in the reaction, we did a control reaction with BSA and found no stimulation of FEN1. In the presence of 5 nM FEN1 and 20 nM BSA only 8% of 1 nM substrate was incised (Figure 2A, lane 9).Figure 2.

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