Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO.
Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.
Orren
D K
DK
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Brosh
R M
RM
Nehlin
J O
JO
Machwe
A
A
Gray
M D
MD
Bohr
V A
VA
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis; D004277:DNA, Single-Stranded / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D000071657:Werner Syndrome Helicase
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D015112:4-Nitroquinoline-1-oxide; D004247:DNA; D005090:Exodeoxyribonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase