Investigations of dynamic, higher-order DNA polymerase complexes in archaea.

As the blueprint for cellular functions, DNA must be properly maintained and copied. Exposure to DNA damaging agents leads to formation of genomic lesions which cause stalling of high-fidelity (HiFi) DNA polymerases (Pols) during replication. However, the damaged DNA template may still be replicated by specialized Pols which perform translesion synthesis (TLS). Though TLS provides a mechanism of cellular tolerance to DNA damage, it also presents the risk of mutagenesis which must be regulated. Recent investigations have characterized a network of protein-protein interactions among multiple Pols and their accessory factors, highlighting several points of TLS Pol regulation to be explored. This collection of work describes the formation of an archaeal supraholoenzyme (SHE) replication complex on DNA, composed of the HiFi PolB1 and TLS PolY bound to the processivity clamp PCNA123. Formation of this higher-order complex is mediated through individual Pol contacts with PCNA123 and direct contacts between the two Pols. This leads to the hypothesis that DNA is ‘handed off’ between Pols within a SHE complex, rather than distributively exchanged. Upon encountering a DNA lesion, PolB1 is stalled and must hand off the DNA for TLS by PolY. After bypass of an 8-oxoguanine lesion by PolY, an intermediate position was identified three base pairs downstream of the lesion. This points to a specific position where PolY passes the DNA back to PolB1 to restore HiFi activity. The coupling of Pols within the SHE complex optimizes their combined activities to promote efficient bypass and extension from the lesion while limiting error-prone TLS activity. This work also reports findings related to alternative higher-order complexes of PolB1, including a homotrimeric PolB1 complex and a heterotrimeric PolB1 associated with two novel binding proteins. Altogether, this work encourages further study of higher-order DNA polymerase complexes in other organisms. By understanding these points of regulation and how DNA is handed off among the various Pols, these mechanisms may be targeted to minimize the adverse effects of TLS.