Impact of ATR's role in translesion synthesis on prevention of DNA damage induced mutagenesis and chromosomal instability - Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Biochemical, structural, and genetic studies have indicated that TLS Pols play highly specialized roles in replicating through DNA lesions. Although there is a great deal of information on the structure and function of TLS Pols, and on the multiplicity of pathways that mediate predominantly error-free TLS and which utilize the action of one TLS Pol or the sequential action of two Pols, there is no information on how these TLS processes are regulated. Based upon the evidence we provide here, we propose a role for ATR kinase in normal human cells in (a) promoting predominantly error-free TLS via multiple pathways; (b) promoting TLS in conjunction with the replisome stalled and stabilized at DNA lesion sites; and (c) in coordinating TLS by the sequential action of two Pols in which one Pol inserts a nucleotide opposite the DNA lesion and another Pol extends synthesis therefrom. In Aim 1, we will carry out studies to establish that unlike in ATR proficient cells where TLS operates via multiple, predominantly error-free pathways, TLS in ATR deficient cells operates via a single highly error-prone pathway. In Aim 2, to establish that ATR promotes proficient TLS dependent replication through DNA lesions in conjunction with the replisome at replication forks (RFs) stalled at DNA lesion sites, we will analyze the ways in which TLS in ATR deficient cells differs from that in ATR proficient cells. For these studies, we will: (a) determine the extent by which RF progression through UV lesions is inhibited in ATR deficient cells compared to that in ATR proficient cells; (b) determine that whereas TLS operates in conjunction with replisome in ATR proficient cells, the replisome disassembles in ATR deficient cells; subsequently, TLS occurs in gaps, requires PrimPol for initiating DNA synthesis, and CRL4Cdt2 ubiquitin ligase for PCNA ubiquitination; (c) determine whether accumulation of Pol at cyclobutane pyrimidine dimers (CPDs) occurs with slower kinetics and at reduced levels in ATR deficient cells compared to that in ATR proficient cells; and (d) determine whether RF collapse resulting from replisome disassembly at DNA lesion sites in ATR deficient cells confers a large elevation in chromosomal aberrations and apoptotic cell death. In Aim 3, we will analyze the role of ATR mediated phosphorylation of TLS Pols in the formation of a TLS Pol ensemble so that the two different steps of TLS dependent upon two Pols occur in one physical entity, rather than independently. The evidence that in contrast to ATR proficient cells, TLS dependent replication through DNA lesions in ATR deficient cells operates in highly mutagenic ways, occurs in gaps, is conducted differently and much less efficiently, and that chromosomal instability and apoptotic cell death are highly elevated in the absence of ATR, will be important for establishing that by restraining DNA damage induced mutagenesis, chromosomal instability, and apoptotic cells death, ATR’s role in TLS would contribute to genome stability and cellular homeostasis.
