Isolation and characterization of DNA damage-recognition proteins expressed in the unicellular alga Chlorella pyrenoidosa and zebrafish (Danio rerio) embryos

• Main Authors: Yi-Show Lai, 賴意繡
• Other Authors: Todd Hsu
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/20839970035886835277

博士 === 國立臺灣海洋大學 === 生物科技研究所 === 93 === Abstract Ultraviolet (UV) light is a component of the solar spectrum and UV penetrating from atmosphere into water is known to damage aquatic organisms present in the water surface layer. At the molecular level, UV irradiation significantly distorts the helical structure of DNA due to the formation of dipyrimidine photoproducts between two adjacent pyrimidines. To avoid producing error-prone replication or transcriptional blockage, UV-damaged DNA in most organisms is repaired primarily by excision-dependent pathways and DNA mismatches are corrected by mismatch repair. DNA repair pathways involving multipolypeptides are generally initiated by the binding of DNA damaged-recognition proteins to lesion sites. The unicellular alga Chlorella pyrenoidosa and zebrafish (Danio rerio) at fast-growing or early developmental stages are selected in this dissertation as the model systems for aquatic organisms to study proteins binding preferentially to DNA mispairs or dipyrimidine photoproducts. A duplex probe carrying a single G-T mispair was revealed by EMSA as a better binding target than those carrying other types of mispair for DNA mismatch recognition proteins in C. pyrenoidosa extracts. Two 13-kDa G-T binding polypeptides possessing pIs of 5.3 and 5.5 were isolated after resolving affinity-captured proteins by 2-D gel electrophoresis and the two factors were found to bind 5.5- and 2.8-fold stronger to heteroduplex than to homoduplex DNA, respectively. No proteins significantly homologous to the two algal G-T binding proteins were found by peptide mass fingerprinting (PMF). The sequence of a peptide generated from trypsin-cleavage of one G-T binding factor (pI 5.5) could be aligned with the amino acid sequences that form the C-terminal active sites of human and mouse mismatch-specific uracil/thymine-DNA glycosylases, suggesting the possibility of this factor as an algae- or a Chlorella-specific DNA mismatch glycosylase. UV-damaged-DNA binding activities in C. pyrenoidosa extracts recognizing both CPDs and 6-4PPs were also detected by EMSA. A 70-kDa polypeptide binding preferentially to a duplex probe carrying CPDs and 6-4PPs was pulled down from the algal extracts by affinity adsorption. Apparently higher levels of three 60 to 62-kDa polypeptides were eluted by 0.12% SDS from UV-irradiated than from unirradiated DNA immobilized on agarose beads when affinity-captured proteins were analyzed by 2-D gel electrophoresis. The 70-kDa polypeptide observed by SDS-PAGE might be an accumulation of these 60 to 62-kDa binding factors. Each SDS-eluted UV-binding polypeptide was found to bind 2 to 5-fold stronger to irradiated than to unirradiated DNA. None of the algal binding factors displayed significant sequence homology with proteins contained in the Mascot data base. Although algal proteins having higher affinities for mismatched or UV-damaged DNA were successfully isolated from C. pyrenoidosa extracts, little progress in protein identification was achieved because of the extremely low homology between algal DNA damage-recognition proteins and proteins already identified. The extracts of zebrafish (Danio rerio) early embryos and early larvae were found to contain different DNA damage-recognition proteins. Immunoblot analysis of proteins captured by agarose beads carrying a UV-irradiated duplex oligonucleotide revealed the presence of XPA in the extracts of 84-hr-old larvae, while no proteins homologous to those associated with human NER could be found in the extracts of 12-hr-old embryos. Incubation of the extracts of zebrafish embryos with a probe containing a 6-4PP produced high-shifting gel retardation complexes and participation of zebrafish vitellogenin 1(Vg1)-like proteins in generating the UV-binding activity was revealed by mass spectrometric analysis of proteins contained in the binding complexes. The binding of 12-hr old zebrafish extracts to 6-4PPs was inhibited to 60% of control by the addition of a rabbit anti-carp Vg antiserum to gel shift assay mixtures. UV-damaged-DNA binding proteins synthesized in zebrafish embryos could be efficiently separated from nonspecific DNA binding proteins by a preparative isoelectrofocusing. Nonspecific binding proteins were collected at pH from 4 to 6, yet the majority of zebrafish proteins binding preferentially to 6-4PPs were found to possess pIs about 7 to 9 and proteins displaying the strongest UV-dependent binding were isoelectrofocused between pH from 7 to 8. Four 25-kDa polypeptides having pIs between 7.3 to 7.8 were thought to be important damage-recognition factors after comparing 2-D polypeptide patterns produced by protein fractions showing strong, moderate, low and no UV-specific binding activity. Although the four polypeptides were not binding targets for a monoclonal anti-zebrafish Vg1 antibody, they all contained amino acid sequences LPIIVTTYAK and IPEITMSK which also appear in the C-terminal Lv2 region of zebrafish Vg1 according to tandem mass spectrometry(MS/MS). One 25-kDa polypeptide possessing a pI about 7.4 was shown to bind strongly to UV-damaged DNA after affinity adsorption of the active protein fraction with a 6-4PP probe and some Lv2 sequences present in other 25-kDa polypeptides were missing in this UV-binding factor. Since several 100 to 25 kDa Vtg1-like proteins produced in 12 to 96-hr-old zebrafish could be detected by the anti-zebrafish Vg1 antibody, the four 25-kDa polypeptides totally unreactive to the same antibody suggested that they originated from a source unrelated to proteolytic degradation of Vg1. The four 25-kDa polypeptides were also identified as phosphoproteins, but not glycoproteins, according to selective protein staining. The original zebrafish Vg1 having a molecular mass of 150 kDa is a metalloprotein whose degradation during developmental process is known to provide amino acids and essential metals for growing embryos. Our results revealed the identification of a group of novel embryonic proteins highly homologous to the Lv2 region of Vg1 and one low-molecular-weight Vg1-like protein might participate in DNA repair or recombination based on its preferential binding to UV-damaged DNA.