Structural And Biochemical Study Of The Key Helicase Enzyme In Double Strand DNA Rapairment System RecF Pathway DrRecQ From Deinococcus Radiodurans

RecQ proteins are DNA unwinding enzyme (helicase) playing critical roles in maintaining of genomic integrity in any organisms ranging from bacteria to human beings.Similar with other DNA helicases, RecQ proteins are ATP-dependent molecular motors that can unwind double-strand (ds) DNA. But, what makes the members of RecQ family different from other helicase is their unusual breadth of activities, including roles in DNA replication, recombination and repair1. In humans, loss of RecQ gene function is associated with cancer predisposition and premature aging. Thus, the RecQ family has attracted considerable interest in recent years due to their role not only in suppression of genome instability, but also in the avoidance of human disease.The first RecQ helicase was identified by Nakayama and Umezu in Escherichia coli where it functions in the RecF recombination pathway through a screening for mutants resistant to thymineless-death2,3. This pathway participates in homologous recombination and repair of ultraviolet light-induced DNA damage4. RecQ and other proteins in the RecF pathway appear to catalyze reactions that help repair replication forks that have stalled at sites of DNA damage5. RecQ genes have been identified in every bacterial and eukaryotic species sequenced to date. Three conserved domains are commonly found in RecQ helicase:helicase, RecQ-C-terminal (RecQ-Ct) and Helicase-and-RNaseD-like-C-terminal (HRDC) sequence elements6. Among them the HRDC domain forms an independent helical bundle and involves in DNA binding, particularly in conferring some degree of DNA substrate specificity7,8.It is well-known that DNA double-strand breaks are considered as the most severe form of genomic damage. D. radiodurans bacterium is among the best-known organisms found to resist extremely high exposures to desiccation and ionizing radiation, both causing extensive DNA double-strand breaks. Thus, its robust radio-resistance makes it an outstanding model system for study of DNA repair system7. In contrast to nearly every other identified RecQ family members, the RecQ helicase from the radio-resistant bacterium D. radiodurans (DrRecQ) encodes three HRDC domains at its C terminus. Unusual structure with a special domain arrangement in the DrRecQ attracts the attention of researchers:the extremely high radioresistance of the bacteria and its great ability to repair damaged DNA possibly can be explained by these extraordinary peculiarities of the protein structure. The complementation and biochemical function of the DrRecQ variants with different domains truncated in vitro suggested that both the helicase and three HRDC domains are necessary for RecQ functions in D. radiodurans, while three HRDC domains have a synergistic effect on the whole function.Here our research is to illustrate the real functions and regulatory mechanism of DrRecQ that plays as the first key protein in RecF pathway which is proved to the main recombinational double strand DNA break repair pathway in D. radiodurans. In this thesis, we concentrated on the structure and biochemical research of DrRecQ full-length proteins and two truncated proteins----DrRecQ1-824(Helicase+RecQ-Ct+3HRDC),DrRecQ1-610(Helicase+RecQ-Ct+HRDC1)andDrRecQ1-520(Helicase+RecQ-Ct).Working as a helicase enzyme, DrRecQ can bind with different kinds of DNA substrates but with unequal binding affinity in the absence of ATP. While mixed with ATP, DrRecQ can unwind double strand DNA and Holiday Junction. Here we use the electrophoretic mobility shift assay (EMSA) of DNA-protein interaction and native gel to detect different binding affinities with DNA substrates and its helicase activity. What is more, we obtained all the crystals of the three proteins.However, a crystal structure of the intact DrRecQ DNA helicase is not available:a long loop between two HRDC domains makes impossible its crystallization and a detailed analysis of the whole structure.Since SAXS is one of the powerful method to examine a structure of biological macromolecules, especially in solution at conditions close to physiological, aims of the present project are (i) a determination of solution structures of the intact DrRecQ, and of the protein with the only HRDC domain,(ii) consistent study of processes of specific interactions of the DrRecQ with various specimens of DNA, and (iii) a construction of adequate structural models based on the synchrotron SAXS data. In the present project a structure of a full-length RecQ helicase from D. radiodurans (DrRecQ1-824) and a structure of the protein with the only HRDC domain (DrRecQ1-610) were studied by small-angle X-ray scattering to determine mutual organization of Helicases and HRDC domains in solution. High-resolution structures of isolated parts of the DrRecQ were used for comparison and structural modeling of the specimen on the base of obtained SAXS data. SAXS modeling revealed more compact structure for the DrRecQ1-824protein by comparison with the DrRecQ1-610one. In solution both proteins demonstrated circular organization of the Helicases with hole in the middle of the structure unlike crystals. From all the experimental data we obtained, we demonstrate that DrRecQ not only can unwind double strand DNA functions in the first step of the RecF pathway but also HRDCs can help DrRecQ have high affinity with Holliday junction. DrRecQ might participate in the resolution or dissolution of double Holliday junctions at the final steps of homologous recombination.