Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer.
We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.
Gupta
Rigu
R
Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Sharma
Sudha
S
Sommers
Joshua A
JA
Jin
Zhe
Z
Cantor
Sharon B
SB
Brosh
Robert M
RM
D050976:Basic-Leucine Zipper Transcription Factors; D001943:Breast Neoplasms / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D051856:Fanconi Anemia Complementation Group Proteins; D006801:Humans; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C102108:BACH1 protein, human; D050976:Basic-Leucine Zipper Transcription Factors; D045566:DNA, Cruciform; D004277:DNA, Single-Stranded; D051856:Fanconi Anemia Complementation Group Proteins; D014157:Transcription Factors; D004247:DNA; D004265:DNA Helicases