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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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Related in: MedlinePlus

Co-localization of endogenous FEN1 and overproduced 3xFlag-RECQL5β-Myc in HeLa cells treated with H2O2. Twenty-four hours after transfection of plasmid encoding 3XFlag-RECQL5β-Myc, non-synchronized cells were incubated in the presence or absence of 100 µM H2O2 for 30 min and then fixed. Cells were triply stained for FEN1 (red), 3xFlag-RECQL5β-Myc (green) and DNA (blue) as described in the ‘Materials and Methods’ section, and analyzed by confocal microscopy. Yellow color in superimposed images (merge) indicates co-localization of FEN1and 3xFlag-RECQL5β-Myc. White bar represents 50 µm.
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Figure 8: Co-localization of endogenous FEN1 and overproduced 3xFlag-RECQL5β-Myc in HeLa cells treated with H2O2. Twenty-four hours after transfection of plasmid encoding 3XFlag-RECQL5β-Myc, non-synchronized cells were incubated in the presence or absence of 100 µM H2O2 for 30 min and then fixed. Cells were triply stained for FEN1 (red), 3xFlag-RECQL5β-Myc (green) and DNA (blue) as described in the ‘Materials and Methods’ section, and analyzed by confocal microscopy. Yellow color in superimposed images (merge) indicates co-localization of FEN1and 3xFlag-RECQL5β-Myc. White bar represents 50 µm.

Mentions: We investigated the subcellular localization of 3XFlag-RECQL5β-Myc and endogenous FEN1 by confocal microscopy to explore whether 3XFlag-RECQL5β-Myc co-localizes with FEN1 before and after DNA damage. We transiently transfected HeLa cells with plasmid encoding a full-length copy of 3XFlag-RECQL5-Myc encoding sequence. Twenty-four hours post transfection, the cells were treated with 100 µM H2O2 for 30 min to introduce oxidative stress and DNA damage. In asynchronous untreated HeLa cells, FEN1 and RECQL5β displayed diffused nuclear staining with some concentrated foci (Figure 8). In H2O2 treated cells, we observed that both FEN1 and RECQL5β formed distinct nucleolar foci and these foci co-localized (Figure 8).Figure 8.


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)

Co-localization of endogenous FEN1 and overproduced 3xFlag-RECQL5β-Myc in HeLa cells treated with H2O2. Twenty-four hours after transfection of plasmid encoding 3XFlag-RECQL5β-Myc, non-synchronized cells were incubated in the presence or absence of 100 µM H2O2 for 30 min and then fixed. Cells were triply stained for FEN1 (red), 3xFlag-RECQL5β-Myc (green) and DNA (blue) as described in the ‘Materials and Methods’ section, and analyzed by confocal microscopy. Yellow color in superimposed images (merge) indicates co-localization of FEN1and 3xFlag-RECQL5β-Myc. White bar represents 50 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875029&req=5

Figure 8: Co-localization of endogenous FEN1 and overproduced 3xFlag-RECQL5β-Myc in HeLa cells treated with H2O2. Twenty-four hours after transfection of plasmid encoding 3XFlag-RECQL5β-Myc, non-synchronized cells were incubated in the presence or absence of 100 µM H2O2 for 30 min and then fixed. Cells were triply stained for FEN1 (red), 3xFlag-RECQL5β-Myc (green) and DNA (blue) as described in the ‘Materials and Methods’ section, and analyzed by confocal microscopy. Yellow color in superimposed images (merge) indicates co-localization of FEN1and 3xFlag-RECQL5β-Myc. White bar represents 50 µm.
Mentions: We investigated the subcellular localization of 3XFlag-RECQL5β-Myc and endogenous FEN1 by confocal microscopy to explore whether 3XFlag-RECQL5β-Myc co-localizes with FEN1 before and after DNA damage. We transiently transfected HeLa cells with plasmid encoding a full-length copy of 3XFlag-RECQL5-Myc encoding sequence. Twenty-four hours post transfection, the cells were treated with 100 µM H2O2 for 30 min to introduce oxidative stress and DNA damage. In asynchronous untreated HeLa cells, FEN1 and RECQL5β displayed diffused nuclear staining with some concentrated foci (Figure 8). In H2O2 treated cells, we observed that both FEN1 and RECQL5β formed distinct nucleolar foci and these foci co-localized (Figure 8).Figure 8.

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