Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand.

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5' overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

Recent Advances in Understanding the Structures of Translesion Synthesis DNA Polymerases.

DNA damage in the template strand causes replication forks to stall because replicative DNA polymerases are unable to efficiently incorporate nucleotides opposite template DNA lesions. To overcome these replication blocks, cells are equipped with multiple translesion synthesis polymerases that have evolved specifically to incorporate nucleotides opposite DNA lesions. Over the past two decades, X-ray crystallography has provided a wealth of information about the structures and mechanisms of translesion synthesis polymerases. This approach, however, has been limited to ground state structures of these polymerases bound to DNA and nucleotide substrates. Three recent methodological developments have extended our understanding of the structures and mechanisms of these polymerases. These include time-lapse X-ray crystallography, which allows one to identify novel reaction intermediates; full-ensemble hybrid methods, which allow one to examine the conformational flexibility of the intrinsically disordered regions of proteins; and cryo-electron microscopy, which allows one to determine the high-resolution structures of larger protein complexes. In this article, we will discuss how these three methodological developments have added to our understanding of the structures and mechanisms of translesion synthesis polymerases.