Structural basis of long-range to short-range synaptic transition in NHEJ.

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.

Redundant but essential functions of PARP1 and PARP2 in DNA ligase I-independent DNA replication.

While DNA ligase I (LigI) joins most Okazaki fragments, a backup pathway involving poly(ADP-ribose) synthesis, XRCC1 and DNA ligase IIIα (LigIIIα) functions along with the LigI-dependent pathway and is also capable of supporting DNA replication in the absence of LigI. Here we have addressed for the first time the roles of PARP1 and PARP2 in this pathway using isogenic null derivatives of mouse CH12F3 cells. While single and double null mutants of the parental cell line and single mutants of LIG1 null cells were viable, loss of both PARP1 and PARP2 was synthetically lethal with LigI deficiency. Thus, PARP1 and PARP2 have a redundant essential role in LigI-deficient cells. Interestingly, higher levels of PARP2 but not PARP1 associated with newly synthesized DNA in the LIG1 null cells and there was a much higher increase in PARP2 chromatin retention in LIG1 null cells incubated with the PARP inhibitor olaparib with this effect occurring independently of PARP1. Together our results suggest that PARP2 plays a major role in specific cell types that are more dependent upon the backup pathway to complete DNA replication and that PARP2 retention at unligated Okazaki fragments likely contributes to the side effects of current clinical PARP inhibitors.

An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex.

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170-755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.

Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV.

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

POLD1 variants leading to reduced polymerase activity can cause hearing loss without syndromic features.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.

Altered DNA ligase activity in human disease.

The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

Repair of DNA double-strand breaks by mammalian alternative end-joining pathways.

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

The SWI/SNF ATP-dependent nucleosome remodeler promotes resection initiation at a DNA double-strand break in yeast.

DNA double-strand breaks (DSBs) are repaired by either the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway. Pathway choice is determined by the generation of 3΄ single-strand DNA overhangs at the break that are initiated by the action of the Mre11-Rad50-Xrs2 (MRX) complex to direct repair toward HR. DSB repair occurs in the context of chromatin, and multiple chromatin regulators have been shown to play important roles in the repair process. We have investigated the role of the SWI/SNF ATP-dependent nucleosome-remodeling complex in the repair of a defined DNA DSB. SWI/SNF was previously shown to regulate presynaptic events in HR, but its function in these events is unknown. We find that in the absence of functional SWI/SNF, the initiation of DNA end resection is significantly delayed. The delay in resection initiation is accompanied by impaired recruitment of MRX to the DSB, and other functions of MRX in HR including the recruitment of long-range resection factors and activation of the DNA damage response are also diminished. These phenotypes are correlated with a delay in the eviction of nucleosomes surrounding the DSB. We propose that SWI/SNF orchestrates the recruitment of a pool of MRX that is specifically dedicated to HR.