Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family.
RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (approximately 59 kDa) N-terminal and a small (approximately 9 kDa) C-terminal domain. A short N-terminal segment (7 or 21 residues) is also shown to be sensitive to proteases. The effects of removing these regions of RecQ are tested in vitro. Removing 21 N-terminal residues from RecQ severely diminishes its DNA-dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays. In contrast, removing the approximately 9 kDa C-terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA. These results establish the biochemical roles of an N-terminal sequence motif in RecQ catalytic function and for the C-terminal RecQ domain in stable DNA binding.
Bernstein
Douglas A
DA
Department of Biomolecular Chemistry, 550 Medical Science Center, 1300 University Avenue, University of Wisconsin, Madison, WI 53706-1532, USA.
Keck
James L
JL
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion
D011994:Recombinant Proteins; D004247:DNA; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases