Three HRDC domains differentially modulate Deinococcus radiodurans RecQ DNA helicase biochemical activity.
RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNA-dependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.
Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706-1532, USA.
Michael P
MP
Killoran
James L
JL
Keck
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry*; D034301:Deinococcus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D053484:RecQ Helicases