Brh2 Promotes a Template-Switching Reaction Enabling Recombinational Bypass of Lesions during DNA Synthesis  Nayef Mazloum, William K. Holloman  Molecular Cell  Volume 36, Issue 4, Pages 620-630 (November 2009) DOI: 10.1016/j.molcel.2009.09.033 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Circumventing a Lesion Blocking Leading-Strand Synthesis A lesion (caret) in the leading strand template causes uncoupling of the replisome so that lagging-strand synthesis continues in the absence of leading-strand synthesis. For synthesis to continue, three general paths can be taken. On the left side, there is fork regression followed by reversal (scheme I) or recombination (scheme II) to reestablish replication after dissolution of the double Holliday junction (dHJ). In the middle is daughter-strand gap repair initiated by single strand invasion (scheme III). On the right side, there is fork cleavage followed by Rad51-mediated 3′ end invasion (scheme IV) or 5′ end invasion (scheme V) to reestablish replication. Holliday junction (HJ) resolution could result in transfer of the lesion to the lagging strand sister chromatid. Parental DNA strands are drawn in gray, and nascent strands are in black. The arrowheads represent 3′ ends of nascent strands. The short arrows represent Okazaki fragments. Molecular Cell 2009 36, 620-630DOI: (10.1016/j.molcel.2009.09.033) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 End Preference in D Loop Formation The schematic depicts the design of reaction in which superhelical plasmid pBS II DNA forms D loops with ss or tailed 32P-labeled (star) oligomers. (A) Reaction mixes contained 32P-labeled ss100-mer (+ strand) that is completely homologous or else homologous at the proximal (5′ hml) or distal (3′ hml) end. D loop formation was initiated using 400 nM Brh2 and/or 800 nM Rad51 as indicated. (B) Reaction mixes contained 32P-labeled 49-mer (− strand) annealed to 100-mer (+ strand) or 32P-labeled 49-mer (+ strand) annealed to 100-mer (− strand) and 400 nM Brh2 and/or 400 nM Rad51 as indicated. In reaction mixes containing RPA, the tailed substrates were precoated with RPA at a ratio of 1 RPA per 6 nucleotides of single-stranded tail. Molecular Cell 2009 36, 620-630DOI: (10.1016/j.molcel.2009.09.033) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Brh2 Promotes Duplexed D Loop Formation with Tailed DNA The schematic depicts the reactions in which 32P-ss100-mer (+ strand) or 32P-labeled 49-mer (−)/ss100-mer (+) 5′ tailed DNA was used as substrate in D loop reactions. After incubation with Rad51 and/or Brh2 as indicated, mixes were split, and one aliquot was digested with restriction endonuclease EcoRI (RE). A representative gel is shown. Results from 6 independent determinations are tabulated graphically beneath with error bars indicating standard deviations. Molecular Cell 2009 36, 620-630DOI: (10.1016/j.molcel.2009.09.033) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Extension of Duplexed D Loops by DNA Synthesis (A) The schematic illustrates the course of reaction in which D loop DNA formed with 32P-ss100-mer (+) or duplexed D loop DNA formed with 32P-labeled 49-mer (-)/ss100-mer (+) 5′ tailed DNA was extended with DNA polymerase (Pol). (B) D loops were formed in the standard reaction with 32P-labeled ss100-mer (+), pBS II DNA, Rad51, and Brh2. Following addition of DNA polymerase and cofactors, reaction was continued for 15 min. Products were analyzed by electrophoresis on a neutral (left panel) or alkaline gel (right panel). (C) Reactions were performed as in (B) but terminated after the times indicated and the products analyzed by electrophoresis on an alkaline gel. (D) Reactions were performed with increasing Rad51: 80 nM (lanes c and h), 160 nM (lanes d and i), 400 nM (lanes e and j), and 800 nM (lanes f and k) with or without 400 nM Brh2 as shown. Alternatively, reactions contained increasing Brh2: 80 nM (lanes n and s), 160 nM (lanes o and t), 400 nM (lanes p and u), and 800 nM (lanes q and v) with or without 800 nM Rad51. In the left panel, reactions contained plasmid DNA and 32P-labeled 49-mer (−)/ss100-mer (+) 5′-tailed DNA as substrates; in the right panel, reactions contained plasmid DNA and 32P-ss100-mer (+). After reaction, DNA polymerase and cofactors were added under standard conditions, and incubation continued for 1 hr. In DNA polymerase reactions with 32P-ss100-mer (+) alone (lanes b and m), there was elongation to yield an almost 2×-length product (hpmer) most likely due to folding back of the ss100-mer on itself to form a hairpin (hp) and then fill-in synthesis. (E) Reactions were performed with Rad51 and Brh2 with 32P-labeled 49-mer (−)/ss100-mer (+) 5′-tailed DNA substrate. Components were deleted as indicated. Following addition of DNA polymerase, the extension reaction was continued for 15 min. Products were analyzed by electrophoresis on a neutral (left panel) or alkaline gel (right panel). Molecular Cell 2009 36, 620-630DOI: (10.1016/j.molcel.2009.09.033) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Lesion Bypass during DNA Synthesis Enabled by Brh2 (A) 5′-tailed DNAs composed of 32P-labeled 49-mer (−) or 24-mer (−) annealed to 100-mer (+) or abasic 100-mer (+) were used in DNA synthesis reactions containing polymerase. The schematic illustrates that DNA synthesis will be aborted by a stretch of three abasic residues located approximately in the middle of the template strand. After reaction, the products were examined on polyacrylamide gels under denaturing conditions. (B) The schematic illustrates that DNA synthesis primed by the 5′-tailed molecule can be extended past the abasic stretch if recombined with a homologous duplex to form a duplexed D loop. (C and D) 5′-tailed DNAs prepared with 32P-labeled 49-mer (−) or 32P-labeled 24-mer (−) strand annealed with 100-mer containing synthesis-blocking abasic stretch as prepared in (A) were used in D loop reactions with Rad51 (800 nM) and Brh2 (400 nM). After reaction, DNA polymerase was added, and the reaction was continued for 30 min. Products were examined on neutral (left panel) and alkaline gels (right panel). (E) Product bands from (C) and (D) were quantified from the digitized images and plotted graphically. Left panel: D loop yields were determined from the neutral gels as the fraction of radio-label in the bands indicated as D loops with respect to the total label in the lane with 5′-tailed substrates prepared using 32P-labeled 49-mer or 24-mer annealed to 100-mer natural sequence (nat) or 100-mer with abasic residues. Right panel: relative levels of extension products were determined from alkaline gels by quantifying the label constituting the smear of products in lanes from reactions with 32P-labeled 49-mer or 24-mer annealed to either 100-mer natural sequence (nat) or 100-mer with abasic residues. Error bars indicate standard deviations calculated from at least three independent determinations. Molecular Cell 2009 36, 620-630DOI: (10.1016/j.molcel.2009.09.033) Copyright © 2009 Elsevier Inc. Terms and Conditions