Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double-stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared with the seven well-recognized conserved helicase motifs, the role of the Q motif is not well known. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion. To examine the roles of the Q motif in the ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant wild type (WT) and mutant (Q23A) proteins were overexpressed and purified from HEK293T cells. The ChlR1-Q23A mutant abolished the helicase activity of ChlR1, and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but displayed normal ATP binding. The Q motif in FANCJ helicase, a ChlR1 homolog, regulates FANCJ’s dimerization, while our size exclusion chromatography (SEC) indicated that the ChlR1 protein functions as a monomer. A thermal shift assay revealed that ChlR1-Q23A has a similar melting point as ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have similar globular structures, although there are some subtle conformational differences between these two proteins. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not in ATP binding.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2016-02-2413 |
| Date | 2016 February 1900 |
| Contributors | Wu, Yuliang |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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