Author: Andrew James ReedPublisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 244
Book Description
Adenosine, guanosine, cytidine, and thymidine nucleotides are the building blocks of life and are arranged in distinct combinations to give unique genomic DNA sequences. These DNA encode for all biological molecules and processes and therefore the preservation of genomic integrity is essential for cell growth and viability. To do so the cell has evolved specialized enzymes for efficient and faithful DNA replication. However, genomic DNA is continually damaged by reactive agents occurring from host processes or those encountered through the environment. The resulting DNA damage can act as mutagens causing errors in DNA replication and ultimately leading to disease states or can cause blocks to DNA replication causing replication fork collapse and potentially cell death. To counteract the onslaught of DNA damage cells have evolved a multitude of DNA damage repair mechanisms that can directly revert damaged DNA bases back to the canonical bases, remove and replace single DNA bases (base excision repair), or remove and resynthesize segments of DNA containing damage (nucleotide excision repair and mismatch repair). Base excision repair (BER) in humans is initiated by a damage specific DNA glycosylase that recognizes and removes a single damaged base from DNA, resulting in a product abasic site. This action is followed by cleavage of the abasic site containing DNA strand by apyrimidinic/apurinic endonuclease 1 generating a single-nucleotide gapped DNA substrate with a deoxyribophosphate-aducted 5'-end (5'-dRP). This gap is then filled and the 5'-dRP removed by DNA polymerase ß (Polß) resulting in a nicked DNA substrate. Finally, this nick is ligated by DNA LigaseIII/XRCC1 to complete repair. Here I have investigated the structure and function of Polß to better define its role in DNA repair. Through time-resolved X-ray crystallography and pre-steady-state gel-based kinetics, I have identified and characterized a third divalent metal ion utilized in the synthesis of DNA, determined the mechanism of nucleotide stereoselectivity, and elucidated distinct mechanistic steps following nucleotide incorporation.
