End-bridging is required for pol mu to efficiently promote repair of noncomplementary ends by nonhomologous end joining.

DNA polymerase mu is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol mu's ability to promote NHEJ of ends with noncomplementary 3' overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol mu in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol mu is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol mu is less able to synthesize across longer gaps, arguing pol mu must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol mu mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol mu activity during NHEJ of noncomplementary ends can thus be primarily linked to pol mu's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction.