1. DNA Repair Synthesis Facilitates RAD52-Mediated Second-End Capture during DSB Repair 
Michael J. McIlwraith, Stephen C. West 
Molecular Cell 
Volume 29, Issue 4, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
In Vitro Reconstitution of Second-End Capture during DSB Repair
(A) Model for DSB repair by HR. The DSB is first resected to produce 3′-extended ssDNA tails. Strand invasion of one resected break (black) into homologous duplex DNA (blue) generates a D loop that can be extended/migrated by polymerase-mediated DNA repair synthesis. The migrating D loop then captures the second end of the break to allow repair DNA synthesis from the second end of the break. Arrowheads indicate 3′ ends. Red lines indicate de novo DNA synthesis.
(B) Experimental design for second-end capture. The D loop contains a 29 nt long primer strand that can be extended (red arrow) through duplex DNA to migrate the D loop and allow capture of the second end. 5′-32P-labels are indicated with asterisks.
(C) Second-end capture reactions. Unlabeled D loops and 32P-labeled tailed DNA (as shown in Figure 1B) were incubated with human polη, polδ, polι, RAD52, and RPA proteins as indicated, and labeled DNA products were analyzed by 8% native PAGE. In the right-hand panel, the omitted component is indicated. Size-marker DNAs were constructed by annealing oligonucleotides of the appropriate length and sequence. The percentage of fully annealed end-capture product is shown below each lane. The 5′-32P-labels and sites of DNA synthesis are indicated in red.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Time Course of Second-End Capture
(A) Second-end capture reactions were carried out as described in Figure 1C legend using RAD52, RPA, and polη. Aliquots were removed at the times indicated, deproteinized, and the DNA products analyzed by 8% native PAGE.
(B) Quantification of second-end capture. Reactions were carried out as described in (A), in the presence (circles) or absence (triangles) of RAD52 protein, and quantified by phosphorimaging. Error bars show standard deviations for three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3 Requirements for Second-End Capture
(A) Second-end capture reactions were carried out as described in Figure 1C legend, except that varying amounts of RAD52 were added, as indicated. A complete reaction was boiled to disrupt annealed complexes (lane h).
(B) Reactions were carried out as in (A) using the indicated proteins.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4 DNA Repair Synthesis at the Captured Second End
(A) Experimental design. The 5′-labeled tailed second end was captured following polymerase-mediated D loop extension in reactions containing RAD52 and RPA, permitting de novo DNA synthesis from the newly captured end (red lines). Oligonucleotide lengths are indicated.
(B) The products from reactions indicated in (A) were analyzed by 8% denaturing PAGE. Reaction components were omitted as indicated. Gel-purified 42-mer, 60-mer, and 70-mer 32P-end-labeled oligonucleotides were used as size markers. The 32P label on the 5′ end of the substrate is indicated with a red asterisk.
(C) End-capture reactions were performed using the indicated polymerases, and the reaction products were analyzed by 10% denaturing PAGE. (Left panel) The first end (D loop primer) was 32P end labeled (asterisks). (Right panel) The second end (tail) was labeled.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions