Figure 2.

AtPolIs performs MMEJ using microhomology sequence longer than 8 nts at high MgCl₂ concentrations. (A) Exonuclease deficient AtPolIs execute MMEJ on a pss-DNA(8) substrate at physiological dNTP concentrations. Reactions incubated in the absence of dNTPs (lanes1 and 11) show the migration o the labeled substrate. MMEJ reactions were incubated using increasing dTTP (panel I) or dATP (panel II) from 0.0037 to 245.76 μM using four-fold increments (lanes 2–10 and 12–20). At low micromolar dNTP concentrations (lanes 2–5 and 12–15) a MMEJ product of 56 nts is observed. In contrast, a band of 57 nts is observed in reactions incubated with dTTP using high nucleotide concentrations and a band of 58 nts is observed in reactions incubated with high concentrations of dATP. (B) Schematic representation of the pssDNAs substrates used for MMEJ assays. Asterisks indicate the position of the 5′-radioactive label. The microhomology lengths (from 0 to 12 nts), the calculated Tm for DNA synapsis formation and the expected lengths of the MMEJ product are indicated at the right. (C) Denaturing sequencing gel showing MMEJ reactions by wild-type (lanes 2–8) and exonuclease deficient (lanes 10–16) AtPolIBs in comparison to reactions incubated with the exonuclease deficient Klenow Fragment of DNAP I (lanes 18–21). Al reactions contained 10 mM MgCl₂ as a cofactor. A control labeled substrate corresponding to pss-DNA(8) is present in lanes 1, 9 and 17. MMEJ products are observed in reactions incubated with AtPolIBs on substrates with microhomologies longer than eight nucleotides (lanes 6, 7, 8, 14, 15, 16). These products correspond to bands of 56, 58 and 98 nts respectively. In contrast, Klenow Fragment is unable to efficiently use MMEJ substrates and only low amounts of products are formed with pssDNAs of 8 and 12 nts (lanes 20 and 21). TT denotes products generated by a predicted terminal transferase activity.