TY - JOUR
T1 - Yeast and Human Translesion DNA Synthesis Polymerases
T2 - Expression, Purification, and Biochemical Characterization
AU - Johnson, Robert
AU - Prakash, Louise
AU - Prakash, Satya
N1 - Funding Information:
This work was supported by National Institutes of Health Grants CA107650, CA094006, and ES012411.
PY - 2006
Y1 - 2006
N2 - The emergence of translesion DNA synthesis (TLS) as a primary mechanism by which eukaryotic cells tolerate DNA damage has led to a large effort to characterize the biochemical properties of the individual DNA polymerases and their roles in promoting replication past DNA lesions. The low-fidelity Y family DNA polymerases constitute a large proportion of TLS polymerases, and four of the five subfamilies of this class of polymerases are represented in eukaryotes. The eukaryotic B family DNA polymerase Polζ also functions in TLS. We have had success in expressing and purifying these TLS polymerases from yeast cells, sometimes in milligram quantities. The purified proteins have been used to determine their ability to synthesize DNA on various modified templates and to analyze the kinetic efficiencies with which bypass occurs. Purified proteins have also been used to determine the X-ray crystal structures of several Y-family DNA polymerases. This chapter describes a general outline of methods used in our laboratory for the expression and purification of these TLS DNA polymerases from yeast cells and for assaying some of their biochemical properties.
AB - The emergence of translesion DNA synthesis (TLS) as a primary mechanism by which eukaryotic cells tolerate DNA damage has led to a large effort to characterize the biochemical properties of the individual DNA polymerases and their roles in promoting replication past DNA lesions. The low-fidelity Y family DNA polymerases constitute a large proportion of TLS polymerases, and four of the five subfamilies of this class of polymerases are represented in eukaryotes. The eukaryotic B family DNA polymerase Polζ also functions in TLS. We have had success in expressing and purifying these TLS polymerases from yeast cells, sometimes in milligram quantities. The purified proteins have been used to determine their ability to synthesize DNA on various modified templates and to analyze the kinetic efficiencies with which bypass occurs. Purified proteins have also been used to determine the X-ray crystal structures of several Y-family DNA polymerases. This chapter describes a general outline of methods used in our laboratory for the expression and purification of these TLS DNA polymerases from yeast cells and for assaying some of their biochemical properties.
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U2 - 10.1016/S0076-6879(06)08024-4
DO - 10.1016/S0076-6879(06)08024-4
M3 - Review article
C2 - 16793382
AN - SCOPUS:33745219847
SN - 0076-6879
VL - 408
SP - 390
EP - 407
JO - Methods in enzymology
JF - Methods in enzymology
ER -