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The Rad6/Rad18-dependent monoubiquitination of PCNA plays a crucial role in regulating replication past DNA damage in eukaryotic cells. We show here that in human cell-free extracts, efficient PCNA monoubiquitination requires both the synthesis of relatively long DNA tracts and polymerase idling or stalling at sites of DNA modification or DNA secondary structures. This dual dependency suggests a dynamic process in which, following initiation, the DNA synthesizing complex undergoes modifications that make it competent as a mediator for the activation of the Rad6/Rad18 pathway.

The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions. Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication. By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer. Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis. Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif. Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.

The biochemistry and genetics of translesion synthesis (TLS) and, as a consequence, of mutagenesis has recently received much attention in view of the discovery of novel DNA polymerases, most of which belong to the Y family. These distributive and low fidelity enzymes assist the progression of the high fidelity replication complex in the bypass of DNA lesions that normally hinder its progression. The present paper extends our previous observation that in Escherichia coli all three SOS-inducible DNA polymerases (Pol II, IV and V) are involved in TLS and mutagenesis [1]. The genetic control of frameshift mutation pathways induced by N-2-acetylaminofluorene (AAF) adducts or by oxidative lesions induced by methylene blue and visible light is investigated. The data show various examples of mutation pathways with an absolute requirement for a specific combination of DNA polymerases and, in contrast, other examples where two DNA polymerases exhibit functional redundancy within the same pathway. We suggest that cells respond to the challenge of replicating DNA templates potentially containing a large diversity of DNA lesions by using a pool of accessory DNA polymerases with relaxed specificities that assist the high fidelity replicase.