Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends.

Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites). At single-strand breaks, 5'-terminal abasic sites are excised by the 5'-deoxyribose-5-phosphate (5'-dRP) lyase activity of DNA polymerase beta (pol beta): here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'-dRP/AP lyase activity.

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks.

Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway.

Template strand scrunching during DNA gap repair synthesis by human polymerase lambda.

Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids.

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda.

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes.

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.

Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining.

The solution structure and dynamics of the BRCT domain from human DNA polymerase mu, implicated in repair of chromosome breaks by nonhomologous end joining (NHEJ), has been determined using NMR methods. BRCT domains are typically involved in protein-protein interactions between factors required for the cellular response to DNA damage. The pol mu BRCT domain is atypical in that, unlike other reported BRCT structures, the pol mu BRCT is neither part of a tandem grouping, nor does it appear to form stable homodimers.

A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining.

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ.

Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases beta and eta in vitro.

DNA polymerases beta and eta are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin-GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated.

Translesion synthesis past platinum DNA adducts by human DNA polymerase mu.

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency.