Importantly in genetic studies Saccharomyces cerevisiae Pif
Importantly, in genetic studies, Saccharomyces cerevisiae Pif1, a member of the SF1 family of helicases, has been implicated in BIR in conjunction with Pol δ (Chung et al., 2010, Saini et al., 2013, Wilson et al., 2013). Using highly purified S. cerevisiae proteins, a system that permits dissection of the mechanistic underpinnings of D-loop-primed DNA synthesis reaction has been developed (Li et al., 2009, Sebesta et al., 2011, Wilson et al., 2013). Using this system, we have shown that Pif1 greatly stimulates Pol δ-mediated DNA extension within the context of the Rad51-made D-loop. Importantly, we have furnished evidence that Pif1 fulfills two distinct functions in the DNA synthesis reaction; namely, (i) it enhances the ability of the polymerase ensemble to catalyze DNA strand displacement synthesis via an interaction with the proliferating cell nuclear antigen (PCNA), the polymerase processivity clamp, and (ii) concomitant with DNA synthesis, Pif1 dissociates the invading strand to establish a migrating DNA bubble structure (Fig. 1) (Wilson et al., 2013). This chapter describes the materials and experimental procedures for reconstituting the repair DNA synthesis reaction using a ssDNA oligonucleotide as the invading strand and supercoiled dsDNA as the information donor. Our method utilizes Rad51, RPA, and Rad54 to generate D-loops and PCNA, the multisubunit PCNA loader UCF 101 factor C (RFC), the trimeric Pol δ and Pif1 helicase in the DNA synthesis phase of the reaction. The methods for product analyses are also described.
Assembling and Analysis of the D-Loop Reaction This protocol describes the procedures for forming the Rad51 presynaptic filament on 32P-labeled 90-mer ssDNA (Fig. 2A) and the generation of D-loops using supercoiled pBluescript plasmid DNA as recipient and the ssDNA-binding protein RPA and the dsDNA translocase Rad54 as accessory factors (Petukhova et al., 1998, Raschle et al., 2004, Sugiyama et al., 1997). Since Rad54 hydrolyzes a large quantity of ATP, an ATP-regenerating system consisting of creatine phosphate (CP) and creatine kinase (CK) should be included to avoid ATP depletion (Petukhova et al., 1998). Note that RPA, but not Rad54, is also needed for the efficiency of the subsequent DNA synthesis reaction (Yuzhakov, Kelman, Hurwitz, & O'Donnell, 1999).
Repair DNA Synthesis Reaction This protocol is for assembling the DNA synthesis reaction using D-loops generated as described in Section 2.3.3. The protein species needed for this reaction are the three-subunit Pol δ, the five-subunit RFC, homotrimeric PCNA, and the Pif1 helicase. As shown in Fig. 2B, Pol δ together with RFC and PCNA can efficiently extend the 32P-labeled invading strand, while the addition of Pif1 leads to a strong stimulation of the DNA synthesis track length and the formation of a migrating DNA bubble structure with a growing 5′ ssDNA tail produced as a result of Pif1-mediated dissociation of the extended invading strand. Methods for DNA synthesis product analysis are described in 4 Analysis of D-Loops and Extended D-Loops., 5 Analysis of DNA Synthesis Within a Migrating D-Loop.
Analysis of D-Loops and Extended D-Loops.
Analysis of DNA Synthesis Within a Migrating D-Loop
Introduction Genomic DNA in all organisms is continuously modified by multiple endogenous and exogenous factors. Some of these modifications are introduced by specialized enzymes and serve for regulation of gene expression, DNA repair, or defense against foreign DNA. Others appear spontaneously or are introduced by genotoxic agents and may impair various genetic processes and lead to genome instability. In particular, many DNA lesions are known to affect replication dramatically, leading to the replication fork stalling and mutagenesis. Most organisms possess specialized DNA polymerases that are able to bypass certain types of lesions but usually have a much lower replication fidelity [1,2]. Transcription can also be impaired by lesions in the template DNA strand, which may lead to stalling of RNA polymerase (RNAP) or to transcriptional mutagenesis, thus producing mutant RNAs and proteins [, , , , ]. At the same time, RNAP can act as a sensor for DNA lesions, by attracting the DNA repair machinery to the damaged template sites during transcription coupled repair (TCR) (reviewed in Ref. ). Furthermore, stalled transcription complexes can severely compromise genome stability by colliding with the replication machinery [9,10]. Bacterial cells contain a single RNAP, and the process of DNA damage recognition and repair should be highly coordinated with transcription to allow efficient gene expression and DNA replication. However, the ability of bacterial RNAP to transcribe damaged DNA templates has not been systematically studied. Only a handful of lesions have been analyzed in the bacterial transcription system in vitro. In particular, it was shown that similarly to DNA polymerases, the abasic site and 8-oxoguanine promote mutagenic insertion of ATP in the RNA transcript, with transient RNAP pausing [, , ]. At the same time, the molecular mechanisms of translesion RNA synthesis remain poorly understood for most types of damaged nucleotides.