The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery.

Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis.

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS.

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three TLS polymerases interact with the β processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also makes a unique secondary contact with the clamp through non-CBM residues.

Replication stalling activates SSB for recruitment of DNA damage tolerance factors.

Translesion synthesis (TLS) polymerases bypass DNA lesions that block replicative polymerases, allowing cells to tolerate DNA damage encountered during replication. It is well known that most bacterial TLS polymerases must interact with the sliding-clamp processivity factor to carry out TLS, but recent work in has revealed that single-stranded DNA-binding protein (SSB) plays a key role in enriching the TLS polymerase Pol IV at stalled replication forks in the presence of DNA damage. It remains unclear how this interaction with SSB enriches Pol IV in a stalling-dependent manner given that SSB is always present at the replication fork.

Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis.

Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer-template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated.

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes.

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner.

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. , many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events.

Single-molecule imaging reveals multiple pathways for the recruitment of translesion polymerases after DNA damage.

Unrepaired DNA lesions are a potent block to replication, leading to replication fork collapse, double-strand DNA breaks, and cell death. Error-prone polymerases overcome this blockade by synthesizing past DNA lesions in a process called translesion synthesis (TLS), but how TLS polymerases gain access to the DNA template remains poorly understood. In this study, we use particle-tracking PALM to image live Escherichia coli cells containing a functional fusion of the endogenous copy of Pol IV to the photoactivatable fluorescent protein PAmCherry.

R6G on graphene: high Raman detection sensitivity, yet decreased Raman cross-section.

Several recent studies have demonstrated the use of single and few-layer graphene as a substrate for the enhancement of Raman scattering by adsorbed molecules in a method termed graphene-enhanced Raman spectroscopy (GERS). Here we determine the resonance Raman scattering cross-section for the dye molecule rhodamine 6G (R6G) adsorbed on bilayer graphene. For the 1650 cm(-1) R6G mode, we obtain a cross-section of 5.