Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A.
The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.
Cui
Sheng
S
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
Klima
Raffaella
R
Ochem
Alex
A
Arosio
Daniele
D
Falaschi
Arturo
A
Vindigni
Alessandro
A
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004265:DNA Helicases / Q000502:physiology*; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D053484:RecQ Helicases; D051716:Replication Protein A
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I