Identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site-directed mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be 87GKVSKRKAV95 present in the C-terminal third of TP2 as a part of an extended NoLS sequence

BREAST CANCER-ASSOCIATED MISSENSE MUTANTS OF THE PALB2 WD40 DOMAIN, WHICH DIRECTLY BINDS RAD51C, RAD51 AND BRCA2, DISRUPT DNA REPAIR

Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN, and RAD51C/FANCO DNA repair genes have an increased life-time risk to develop breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutations of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I, and L1143P missense mutants/variants of PALB2 WD40 domain are associated with altered direct binding patterns to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, while the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway

Cloning of cDNA Encoding Rat Spermatidal Protein TP2 and Expression in Escherichia coli

Rat spermatidal protein TP2 is a basic nuclear protein containing two atoms of zinc bound per molecule. We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method. The nucleotide sequence of cloned TP2 cDNA differs at a few positions from the sequence already reported in the literature. We have cloned rat TP2 cDNA into the expression vector pTrc 99A. Upon induction with 1 mM IPTG, there was a low level of expression of TP2 which could be recovered in the soluble form. Recombinant TP2 was purified from the soluble extract of E. coli using nickel-agarose and heparin-agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive Zn-65-blottin

Generation of multiple site-specific mutations in a single polymerase chain reaction product

Site-directed mutagenesis has been very valuable in studying the structure–function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2–13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure–function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis

Cloning of cDNA encoding rat spermatidal protein TP2 and expression in Escherichia coli

Rat spermatidal protein TP2 is a basic nuclear protein containing two atoms of zinc bound per molecule. We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method. The nucleotide sequence of cloned TP2 cDNA differs at a few positions from the sequence already reported in the literature. We have cloned rat TP2 cDNA into the expression vector pTrc 99A. Upon induction with 1 mMIPTG, there was a low level of expression of TP2 which could be recovered in the soluble form. Recombinant TP2 was purified from the soluble extract of E. coliusing nickel- agarose and heparin - agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive <SUP>65</SUP>Zn-blotting

Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5' UTR

We have recently reported the cDNA cloning of rat spermatidal protein TP2 and its expression in Escherichia coli using pTrc 99A as the expression vector. However, the expression level was very low. We have now improved the expression of TP2 over fivefold by (1) optimizing the codons for lysine, arginine, proline, leucine, glycine, valine, threonine, alanine, and tyrosine and (2) by engineering the vector-encoded 5' UTR. The expressed protein was in the soluble phase and could be purified to homogeneity by successive chromatography on Zinc-NTA-agarose affinity matrix and heparin agarose. Serendipitously, we have also observed a concomitant hyperinduction of vector encoded β-lactamase gene along with TP2 in the E. coli BL21 (DE3) cells

Generation of multiple site-specific mutations in a single polymerase chain reaction product

Site-directed mutagenesis has been very valuable in studying the structure-function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2-13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure-function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis