Regulation of Metnase’s TIR binding activity by its binding partner, Pso4

Metnase (also known as SETMAR) is a SET and transposase fusion protein in humans and plays a positive role in double-strand break (DSB) repair. While the SET domain possesses histone lysine methyltransferase activity, the transposase domain is responsible for 5′-terminal inverted repeat (TIR)-specific binding, DNA looping, and DNA cleavage activities. We recently demonstrated that human homolog of Pso4 (hPso4) is a Metnase binding partner that mediates Metnase binding to non-TIR DNA such as DNA damage sites. Here we show that Metnase functions as a dimer in its TIR binding. While both Metnase and hPso4 can independently interact with TIR DNA, Metnase’s DNA binding activity is not required for formation of the Metnase-hPso4-DNA complex. A further stoichiometric analysis indicated that only one protein is involved in interaction with dsDNA when Metnase-hPso4 forms a stable complex. Interaction of the Metnase-hPso4 complex with TIR DNA was competitively inhibited by both TIR and non-TIR DNA, suggesting that hPso4 is solely responsible for binding to DNA in the Metnase-hPso4-DNA complex. Together, our study suggests that hPso4, once it forms a complex with Metnase, negatively regulates Metnase’s TIR binding activity, which is perhaps necessary for Metnase localization at non-TIR sites such as DSBs.     

Metnase/SETMAR: a domesticated primate transposase that enhances DNA repair,replication, and decatenation

Metnase is a fusion gene comprising a SET histone methyl transferase domain and a transposase domain derived from the Mariner transposase. This fusion gene appeared first in anthropoid primates. Because of its biochemical activities, both histone (protein) methylase and endonuclease, we termed the protein Metnase (also called SETMAR). Metnase methylates histone H3 lysine 36 (H3K36), improves the integration of foreign DNA, and enhances DNA double-strand break (DSB) repair by the non-homologous end joining (NHEJ) pathway, potentially dependent on its interaction with DNA Ligase IV. Metnase interacts with PCNA and enhances replication fork restart after stalling. Metnase also interacts with and stimulates TopoIIα-dependent chromosome decatenation and regulates cellular sensitivity to topoisomerase inhibitors used as cancer chemotherapeutics. Metnase has DNA nicking and endonuclease activity that linearizes but does not degrade supercoiled plasmids. Metnase has many but not all of the properties of a transposase, including Terminal Inverted Repeat (TIR) sequence-specific DNA binding, DNA looping, paired end complex formation, and cleavage of the 5′ end of a TIR, but it cannot efficiently complete transposition reactions. Interestingly, Metnase suppresses chromosomal translocations. It has been hypothesized that transposase activity would be deleterious in primates because unregulated DNA movement would predispose to malignancy. Metnase may have been selected for in primates because of its DNA repair and translocation suppression activities. Thus, its transposase activities may have been subverted to prevent deleterious DNA movement.     

The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining

Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of γ-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.     

The DNA repair component Metnase regulates Chk1 stability

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.     

The SET Domain Is Essential for Metnase Functions in Replication Restart and the 5’ End of SS-Overhang Cleavage

Metnase (also known as SETMAR) is a chimeric SET-transposase protein that plays essential role(s) in non-homologous end joining (NHEJ) repair and replication fork restart. Although the SET domain possesses histone H3 lysine 36 dimethylation (H3K36me2) activity associated with an improved association of early repair components for NHEJ, its role in replication restart is less clear. Here we show that the SET domain is necessary for the recovery from DNA damage at the replication forks following hydroxyurea (HU) treatment. Cells overexpressing the SET deletion mutant caused a delay in fork restart after HU release. Our In vitro study revealed that the SET domain but not the H3K36me2 activity is required for the 5’ end of ss-overhang cleavage with fork and non-fork DNA without affecting the Metnase-DNA interaction. Together, our results suggest that the Metnase SET domain has a positive role in restart of replication fork and the 5’ end of ss-overhang cleavage, providing a new insight into the functional interaction of the SET and the transposase domains.     

Biochemical characterization of a SET and transposase fusion protein, Metnase: its DNA binding and DNA cleavage activity

Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.     

The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIalpha decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIalpha inhibitors.     

Biochemical characterization of metnase's endonuclease activity and its role in NHEJ repair

Metnase (SETMAR) is a SET-transposase fusion protein that promotes nonhomologous end joining (NHEJ) repair in humans. Although both SET and the transposase domains were necessary for its function in DSB repair, it is not clear what specific role Metnase plays in the NHEJ. In this study, we show that Metnase possesses a unique endonuclease activity that preferentially acts on ssDNA and ssDNA-overhang of a partial duplex DNA. Cell extracts lacking Metnase poorly supported DNA end joining, and addition of wt-Metnase to cell extracts lacking Metnase markedly stimulated DNA end joining, while a mutant (D483A) lacking endonuclease activity did not. Given that Metnase overexpression enhanced DNA end processing in vitro, our finding suggests a role for Metnase's endonuclease activity in promoting the joining of noncompatible ends.     

Expression levels of the human DNA repair protein metnase influence lentiviral genomic integration

We recently identified a Transposase domain protein called Metnase, which assists in repairing DNA double-strand breaks (DSB) via non-homologous end-joining (NHEJ), and is important for foreign DNA integration into a host cell genome. Since integration is essential for productive lentiviral infection we examined whether Metnase expression levels could have an influence on lentiviral genomic integration. Using cells stably transduced to either over- or under-express Metnase we determined that the expression level of Metnase did indeed correlate with live lentiviral integration. Changes in Metnase levels were accompanied by changes in the number of copies of integrated lentiviral cDNA. While Metnase levels affected lentiviral integration, it had no effect on the amount of either total cellular viral RNA, cDNA or 2-LTR circles. Therefore, Metnase enhances the integration of lentivirus DNA into the host cell genome.     

The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN⁶¹⁰ with either DDD⁶¹⁰ or DDE⁶¹⁰ significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN⁶¹⁰ → DDD⁶¹⁰, which restores the ancestral catalytic site, results in loss of function in Metnase.     

The histone methyltransferase SET8 is required for S-phase progression

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.     

The ancient mariner sails again: transposition of the human Hsmar1 element by a reconstructed transposase and activities of the SETMAR protein on transposon ends

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage approximately 50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution.     

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.

In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation. Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease     

Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites

Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.     

NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination

Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.     

Recruitment of the type B histone acetyltransferase Hat1p to chromatin is linked to DNA double-strand breaks

Type B histone acetyltransferases are thought to catalyze the acetylation of the NH₂-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH₂-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.     

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.