Figure 5

AtPrimPol performs microhomology-mediated end-joining and moderated strand-displacement. Reactions were incubated at 10 (Strand displacement assay), 20 (strand displacement assay) 30 min (microhomology assay). All reactions were loaded onto 17% polyacrylamide denaturing gel and analyzed by phosphorimaging. (A) AtPrimPol at a concentration of 400 nM was able to generate intermediate products (lanes 2, 5, 8, and 11) into the gap created during synapse formation between pssDNAs with different length of microhomology sequence (2, 4, 6 and 8 nts), in contrast with the deficient Zn⁺⁺ finger mutant of AtPrimPol, AtPPolΔZF, that improved the primer extension at the same concentration (lanes 3, 6, 9, and 12). As a control of microhomology, reaction containing 100 nM of AtPolIB was capable to fill the gap and exhibited strand-displacement activity, showing a final product up to 57 nts (Lane 13). (B) Strand-displacement on gapped substrates, AtPrimPol fills a 1-nt gap but is unable to displace the blocking oligonucleotide. In contrast, AtPPolΔZf fills the 1-nt gap and displaces the blocking oligonucleotide with minimal efficiency (lanes 2 to 5). A similar pattern is observed on substrates with a 6-nt gap (lanes 8 to 11) or a 16-nt gap (lanes 14 to 17). An exonuclease deficient version of AtPolIB was used as a positive control for strand displacement (Lanes 6, 7, 12,13, 18, and 19). (C) MMEJ activities on, ssDNA. Both AtPrimPol (Lane 2) and of AtPPolΔZF (Lane 3) execute MMEJ on 3′ overhang from ssDNA with 8 nts as microhomology sequence (5′-CCCCGGGG-3′) and produce the 57 nts product as AtPolIB. (D) In presence of an increasing concentration of single stranded DNA binding proteins from A. thaliana, the microhomology reaction using ssDNA as substrate was not affected by AtmtSSB1 or AtmtSSB2 and severely hampered by AtOSB2 and AtWhy2. Original data used to compose this figure is present in Fig. S11.