DNA polymerase (Pol) , a B-family Pol, plays an important role in replication of damaged DNA. Extensive biochemical studies with Pol have shown that it is very inefficient at inserting nucleotides (nts) opposite DNA lesions but is highly efficient at extending synthesis from the correct or incorrect nt opposite DNA lesions. Since translesion DNA synthesis (TLS) through a large variety of DNA lesions requires the sequential action of an inserter and an extender Pol, by extending synthesis opposite from diverse DNA lesions, Pol performs a critical task in the replication of damaged DNA. In yeast, Rev1 performs an indispensable but non-catalytic role as a component of Pol and it increases Pol’s efficiency for extension from incorrect nts opposite DNA lesions. Consequently, the Rev1/Pol complex promotes highly error-prone TLS and thereby accounts for damage induced mutagenesis in yeast. In striking contrast to the requirement of Rev1 for yeast Pol function, Rev1 is not required for Pol function in TLS in normal human cells. Instead, we provide evidence here that Pol acts as an indispensable component of human (h) Pol and in concert with Pol, hPol promotes a predominantly error-free mode of TLS opposite various DNA lesions. In the proposed studies, we will utilize a combination of genetic, cellular, biochemical, and structural studies to: elucidate the role of Pol in Pol dependent TLS in human cells, determine the fidelity and action mechanism of Pol in TLS opposite DNA lesions which impair Watson-Crick (W-C) base pairing, and define the molecular mechanisms that allow Pol’s active site to handle such DNA lesions. In Aim 1, we will: (a) analyze the requirement of Pol and its domains for Pol-dependent TLS and mutagenesis opposite a variety of DNA lesions in human cells; (b) examine the role of Pol in UV induced mutagenesis in the cII gene carried in the mouse genome; (c) determine the requirement of Pol domains for localization of Polinto replication foci in UV damaged human cells; (d) examine whether Pol associates with hPol in a physical complex in UV damaged human cells; and (e) determine the effects of Pol on fork progression in UV irradiated human cells. In Aim 2, we will (a) use steady-state kinetic analyses to determine the catalytic efficiency and fidelity of Pol in inserting nucleotides opposite N1-methyladenine (N1-MeA) and the 3’T and 5’T of a (6-4) TT photoproduct, and (b) carry out pre- steady-state kinetic studies to determine the action mechanism of Pol for inserting the correct nt opposite these DNA lesions which impair W-C base pairing. In Aim 3, we will (a) determine the structures of binary and ternary complexes of Pol bound to N1-MeA template in the absence or presence of an incoming dTTP, respectively, (b) determine the structures of binary and ternary complexes of Pol bound to the (6-4) TT photoproduct-containing templates in the absence or presence of an incoming dATP, respectively, and (c) carry o...