Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3 0 overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.

Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

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Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol switches to dNTP-stabilized misalignment. pol ␤ generates ؊1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.Pre-steady state kinetic studies of DNA polymerase fidelity have been focused on base substitution mutagenesis mechanisms (1, 2). Simple frameshift mechanisms have not yet been addressed despite the destructive biological consequences of having one or a few bases deleted or added. Small frameshifts, predominantly one-base deletions, are made on undamaged DNA by human DNA pol 1 , pol , pol ␤ (to a lesser extent) (3-7), and Escherichia coli pol IV (called simply "pol IV" throughout) (8 -10). Three models have been proposed to explain Ϫ1 frameshifts, namely the classical Streisinger model (11), direct misincorporation misalignment (3, 12), and dNTPstabilized misalignment (13, 14) (Fig. 1).Streisinger slippage results in simple deletions by displacement, i.e. the "looping out" of one or more bases as a primer strand slides along a run of reiterated template bases during replication (Fig. 1). Misincorporation misalignment occurs when DNA polymerase initially forms a mismatched base pair at the 3Ј-primer end that subsequently realigns by pairing with a complementary downstream template base prior to undergoing further extension (Fig. 1). Alternatively, DNA misalignment could occur as the first step followed by the "correct" incorporation of an incoming dNTP opposite a complementary downstream template base, a process referred to as dNTPstabilized misalignment (Fig. 1), which has been observed in the crystal structure of the pol IV homolog Sulfolobus solfataricus Dpo4 in ternary complex with DNA and an incoming nucleotide (15). The bottom line is that all three processes can follow different paths to arrive at the same mutational end point, a Ϫ1 deletion. Determining precise frameshifting mechanisms for individual DNA polymerases during replication and repair is an essential step toward understanding the basic principles of mutagenesis.In this study we perform pre-steady state fluore...