The hSNM1 protein is a DNA 5'-exonuclease

The human SNM1 protein is a member of a highly conserved group of proteins catalyzing the hydrolysis of nucleic acid substrates. Although overproduction is unstable in mammalian cells, we have overproduced a recombinant hSNM1 protein in an insect cell system. The protein is a single-strand 5′-exonuclease, like its yeast homolog. The enzyme utilizes either DNA or RNA substrates, requires a 5′-phosphate moiety, shows very little activity on double-strand substrates, and functions at a size consistent with a monomer. The exonuclease activity requires the conserved β-lactamase domain; site-directed mutagenesis of a conserved aspartate inactivates the exonuclease.     

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.     

A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family

The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.     

DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.     

A Novel Single-Strand Specific 3′–5′ Exonuclease Found in the Hyperthermophilic Archaeon,Pyrococcus furiosus

Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 3′–5′ exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed single-strand specific 3′–5′ exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 3′ to 5′ direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 3′–5′ exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus

The human Werner syndrome protein, WRN, is a member of the RecQ helicase family and contains 3′→5′ helicase and 3′→5′ exonuclease activities. Recently, we showed that the exonuclease activity of WRN is greatly stimulated by the human Ku heterodimer protein. We have now mapped this interaction physically and functionally. The Ku70 subunit specifically interacts with the N-terminus (amino acids 1–368) of WRN, while the Ku80 subunit interacts with its C-terminus (amino acids 940– 1432). Binding between Ku70 and the N-terminus of WRN (amino acids 1–368) is sufficient for stimulation of WRN exonuclease activity. A mutant Ku heterodimer of full-length Ku80 and truncated Ku70 (amino acids 430–542) interacts with C-WRN but not with N-WRN and cannot stimulate WRN exonuclease activity. This emphasizes the functional significance of the interaction between the N-terminus of WRN and Ku70. The interaction between Ku80 and the C-terminus of WRN may modulate some other, as yet unknown, function. The strong interaction between Ku and WRN suggests that these two proteins function together in one or more pathways of DNA metabolism.     

The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ exonuclease for a phosphate moiety at 5′ dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5′-phosphate is due to the formation of inert enzyme–substrate complexes. By examining a λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.