Cytarabine (?-D-arabinofuranosyl cytosine, araC) has been used for the treatment of acute myelogenous leukemia (AML) for over 40 years. AraCTP competes with dCTP for incorporation into DNA; thus, the chemotherapeutic action of araC derives from its ability to inhibit DNA replication. Although the replicative polymerases (Pols) ca insert araCTP at the 3' terminus of newly synthesized DNA, they are inhibited at extending from it. However, human cells harbor a number of translesion synthesis (TLS) DNA Pols that can, in principle, overcome the inhibitory effects of araC on DNA replication by both extending DNA synthesis from araC-terminated 3' ends and by replicating through the araC lesion that becomes incorporated into the template strand. To understand the relative significance and mechanisms of TLS Pols in promoting replication of araC-damaged DNA, we will carry out a combination of genetic, cellular, biochemical, and structural studies.
In Aim 1, we will (a) analyze the roles of TLS Pols in mediating replication through araC in human cells and determine whether they act in an error-free or mutagenic manner; (b) examine the effects of depletions of TLS Pols required for the replication of araC-damaged DNA on the progression of the replication fork in araC treated human cells; and (c) examine the effects of depletions of TLS Pols on the survival of human cells treated with araC.
In Aim 2, we will carry out steady- state kinetic analyses to (a) determine the catalytic efficiencies of TLS Pols for extending from araC at the 3' primer terminus; (b) determine the proficiency and fidelity of TLS Pols for nucleotide (nt) incorporation opposite araC; and (c) analyze the proficiency of TLS Pols for extending from the nt inserted opposite araC; in addition, (d) we will carry out pre-steady state kinetic studies to gain a better understanding of the mechanisms of TLS Pols in extending from araC and in inserting nts opposite araC.
In Aim 3, we will determine ternary complex crystal structures of TLS Pols that (a) insert nts opposite araC in the templating strand; (b) that function in the extension step of TLS by inserting the correct or incorrect nt when araC is paired to the primer terminus; and (c) that are required for the extension of DNA synthesis from araC terminated DNA. Altogether, these studies will provide a deeper understanding of the roles that different TLS Pols play in promoting the replication of araC damaged DNA, and how the TLS Pols manage to accommodate araC into their active sites and their kinetic mechanisms of action. In addition to novel mechanistic information on the roles of TLS Pols in the replication of araC damaged DNA, these studies may posit ways to increase the effectiveness of araC chemotherapy for the treatment of a cancer where relapse is a major problem.
Cytarabine (araC) is the drug of choice for the treatment of acute myeloid leukemia (AML). Although complete remission occurs in a large proportion of patients, relapse occurs in almost all these cases. The proposed studies are important for understanding how human cells surmount the cytotoxic effects of araC and for identifying ways of enhancing the efficacy of araC for the treatment of AML.
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