Error Prone vs Error Free DNA Replication in Human Cells

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The overall goals of the proposed studies are: to examine in human cells the mechanisms involved in "translesion synthesis"(TLS), i.e., the process by which human cells insert nucleotides opposite lesions in the DNA template that block replication by the major replication complex; to examine the mechanisms involved in "damage avoidance", a broad term that refers to various error-free processes by which human cells complete replication without using the damaged DNA as a template; and to determine whether there is a reciprocal relationship between these two alternative pathways. 1.) We will test the hypothesis that when replication by the normal replication complex is blocked by lesions, TLS, using the damaged DNA as a template, involves the products of several specialized human polymerases and the chance that a specific polymerase will introduce "spontaneous" or mutagen-induced mutations during TLS depends upon: a) the nature of the lesion; b) whether it is located in the template strand for leading or lagging strand synthesis; c) the surrounding sequence; and d) the availability of the other competing specialized polymerases in the cell; 2.) We will test the hypothesis that when DNA replication is blocked, human fibroblasts obtain the necessary genetic information from an undamaged homologous copy of the DNA, e.g., the newly- replicated daughter-strand of the corresponding DNA, i.e., the sister chromatid or the corresponding allele (recombination, gene conversion); 3.) We will test the hypothesis that if such "damage avoidance" pathways for obtaining the genetic information from homologous copies of DNA are blocked, use of mutagenic TLS will increase correspondingly, i.e., the relationship is reciprocal; 4.) We will determine a) the nature of the "damage-avoidance" mechanism(s) used to complete lesion-blocked DNA replication b) the relative frequency at which cells use those pathways rather than TLS pathways, and c) the effect of lack of particular gene products on the (reciprocal) relationship between the pathways, using substrates designed to reveal the mechanisms used, from the nature of the products.