Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.
Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.
Kuusk
Silja
S
Department of General and Microbial Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Tartu 51010, Estonia.
Sedman
Tiina
T
Jõers
Priit
P
Sedman
Juhan
J
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000502:physiology*; D004272:DNA, Mitochondrial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D013379:Substrate Specificity; D013997:Time Factors
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D024101:Mitochondrial Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C120692:HMI1 protein, S cerevisiae; D005990:Glycerol