Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination

Stalled replication forks pose a serious threat to genome integrity. To overcome the catastrophic consequences associated with fork demise, translesion synthesis (TLS) polymerases such as poleta promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. By using fractionated cell extracts and purified recombinant proteins, we show that poleta extends DNA synthesis from D loop recombination intermediates in which an invading strand serves as the primer. Extracts from XP-V cells, which are defective in poleta, exhibit severely reduced D loop extension activity. The D loop extension activity of poleta is unusual, as this reaction cannot be promoted by the replicative DNA polymerase delta or by other TLS polymerases such as poliota. Moreover, we find that poleta interacts with RAD51 recombinase and RAD51 stimulates poleta-mediated D loop extension. Our results indicate a dual function for poleta at stalled replication forks: the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.     

Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.     

Escherichia coli RecA promotes strand invasion with cisplatin-damaged DNA

The antitumor drug cisplatin causes intrastrand cross-linking of adjacent guanine residues that severely distorts the DNA backbone. These DNA adducts impede the progress of the replisome and may result in replication fork arrest. In Escherichia coli, the response to cisplatin involves the action of the prototypic recombinase RecA. Here we show that RecA can utilize, albeit at reduced levels, oligonucleotides that bear site-specific cisplatin-induced 1,2 d(GpG) intrastrand cross-links in strand invasion reactions. Binding of RecA to cisplatin-damaged oligonucleotides was not affected, indicating that the impediment was in the pairing step. The cognate E. coli single-strand DNA-binding protein specifically stimulated strand invasion particularly with cisplatin-damaged DNA. These results indicate that RecA is capable of processing the major cisplatin-induced lesion via a recombination mechanism.     

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding.

The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

Human homologue of ariadne promotes the ubiquitylation of translation initiation factor 4E homologous protein, 4EHP

Human homologue of Drosophila ariadne (HHARI) is a RING-IBR-RING domain protein identified through its ability to bind the human ubiquitin-conjugating enzyme, UbcH7. We now demonstrate that HHARI also interacts with the eukaryotic mRNA cap binding protein, translation initiation factor 4E homologous protein (4EHP), via the N-terminal RING1 finger of HHARI. HHARI, 4EHP and UbcH7 do not form a stable heterotrimeric complex as 4EHP cannot immunoprecipitate UbcH7 even in the presence of HHARI. Overexpression of 4EHP and HHARI in mammalian cells leads to polyubiquitylation of 4EHP. By contrast, HHARI does not promote its own autoubiquitylation. Thus, by promoting the ubiquitin-mediated degradation of 4EHP, HHARI may have a role in both protein degradation and protein translation.     

A 35-kDa protein binding to a cytosine-rich strand of hypervariable minisatellite DNA.

A minisatellite-binding protein, Msbp-4, with a molecular mass of 35 kDa has been purified from mouse tumor cells that binds to hypervariable Pc-1 and Pc-2 minisatellites. The binding is much more efficient than that to genetically stable minisatellite homologues. As assayed by Southwestern analysis, Msbp-4 favors multiple copies of the Pc-2 repeat sequence GGCAGGA and requires the cytosine-rich single strand for the binding. The activity is also present in extracts from mouse testis but not from liver. The phosphatase treatment revealed that Msbp-4 is phosphorylated and may have a regulatory function, because dephosphorylation affects the activity and specificity of the binding. Sequence preference is demonstrated by a competition experiment using single-base substitution mutants. Thus, the binding properties of Msbp-4 observed here lead to an implication that the protein-DNA complexes result in formation of a single-stranded DNA loop of the G-rich strand in the minisatellite which may enhance the ability of the minisatellite to undergo recombination.     

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.     

HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein

The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.     

RecA protein promotes strand exchange with DNA substrates containing isoguanine and 5-methyl isocytosine

The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange in vitro. We have examined DNA strand exchange between a 70 nucleotide ssDNA fragment and a 40 bp duplex, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the nonstandard nucleosides 2'-deoxyisoguanosine (iG) and 2'-deoxy-5-methylisocytidine (MiC), respectively. We demonstrate that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and MiC in a genetic code. Experiments of this type also provide another avenue for exploring RecA-facilitated DNA pairing mechanisms.     

Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing

Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.     

The herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) promotes strand invasion

ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.     

A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor

We have examined the supercoiling requirement for the in vitro Mu DNA strand transfer reaction and found that optimal efficiency requires a high level (sigma = -0.06) of donor plasmid superhelicity. At in vivo levels of supercoiling (sigma = -0.025) the reaction does not occur. Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichia coli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%. This protein, which we named supercoiling relief factor was purified to near homogeneity and found to be identical to integration host factor (IHF), a protein known to induce site specific bends in DNA. The dramatic reduction in the supercoiling requirement was promoted by about 1.5 IHF dimers/donor substrate molecule. At these low levels of IHF, the HU requirement for the reaction was also reduced; a synergistic effect of the two proteins resulted in a greater than 10-fold stimulation of the reaction under appropriate conditions. Furthermore, at high concentrations of IHF, HU could be completely eliminated from the reaction.     

RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange

In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.     

The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange

The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.     

Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli

The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.