A. Msh2-Msh3 protection pattern on a 5’ flap substrate (Top) and nicked bubble substrate (Bottom) resulting from OP-Cu cleavage analysis (see , ). Circles: protection (<50% of no protein control), Triangles: enhanced cleavage (>150% of no protein control). B. Rad27 cleavage assay on substrates with various length -5’ flaps (5, 10, 20, and 30 nt). Substrates were incubated with Rad27 (2.5nM) and increasing concentrations of Msh2-Msh3 (0, 12.5 and 25nM). Top: substrate illustration with asterisk indicating site of ³²P label. C. Msh2-Msh3 competes with Rad27 for binding at the base of a 5’ flap. Substrate binding efficiency was studies using electrophoresis mobility shift assay (EMSA). 10 and 20 nt flap substrates were pre-incubated with 2.5nM Rad27 for 10min before adding increasing concentrations of Msh2-Msh3 (0, 2.5, 12.5, 25, 37.5nM). The labeled substrate is illustrated above with the asterisk indicating the site of the ³²P label. The complexes formed are indicated beside the gel, which correspond to the particular band shift. Bottom: illustration of Msh2-Msh3 inhibition of Rad27 cleavage. D. Ligation assay on substrates containing a 12nt unannealed bubble region separated from a nick by a various length annealed DNA regions (2, 4, 6nt). Substrates were incubated with Cdc9 (12.5nM) and increasing concentrations of Msh2-Msh3 (0, 2.5, 5, 12.5, 25, 37.5nM). Top: substrate illustration with asterisk indicating site of ³²P label. Bottom: illustration of Msh2-Msh3 inhibition of Cdc9.

A. Expansion assay on an equilibrating flap substrate with each flap composed of either ten CTG repeats or thirty randomly generated nucleotides. Substrates were incubated with Rad27 (2.5nM), Cdc9 (37.5nM), and increasing concentrations of Msh2-Msh3 (0, 5, 12.5, 25, 37.5nM or 0, 12.5, 25, 27.5nM). Top: substrate equilibrating conformations illustration with asterisk indicating site of ³²P label, TNRs in gray. See also . B. Illustration of Rad27 cleavage intermediates for equilibrating flap substrate. C. Expansion assay on an equilibrating flap substrate containing equilibrating flaps composed of three CTG repeats. Substrates were incubated with Rad27 (2.5nM), Cdc9 (37.5nM), and increasing concentrations of Msh2-Msh3 (2.5, 5, 12.5, 25, 37.5 nM). Top: substrate equilibrating conformations illustration with asterisk indicating site of ³²P label, TNRs in gray.

During Okazaki fragment maturation, the polymerase (pol δ) reaches the 5’ end of the adjacent Okazaki fragment and causes strand displacement. At TNRs (gray), the polymerase dissociates from the DNA and allows for the newly synthesized strand and displaced 5’ flap to compete for annealing to the template. The DNA equilibrates into multiple structural conformations during this process. Left: The upstream newly synthesized flap anneals completely to the template. Msh2-Msh3 binds at the base of the 5’ flap and inhibits Rad27 and Cdc9. Right: Msh2-Msh3 binds and stabilizes loops of various lengths within the two equilibrating flaps. This causes the 5’ flap to partially anneal to the template and create various length flaps. Rad27 and Cdc9 process these flaps to create expanded sequences of various lengths.