Characterisation Of DNA Llgase And Pairing Activities From A Partially Purified Fraction From Rat Testis
Knowledge of the mechanisms of homologous recombination has come from extensive genetic studies on fungi. These studies have led to the formulation of various models which explain the genetic observations. Analysis of the fate of transfected DNA molecules into cells have revealed the existence of additional recombinational pathways in eukaryotes. The biochemistry of homologous recombination has mainly focused on the isolation and characterisation of the recombinase activity from various systems. The E. cold RecA protein remains the best characterised recombinase or pairing protein till date and its reactions in vitro, in particular the strand-transfer reaction, have laid the framework for isolating such proteins from eukaryotic systems. However, limited success has been achieved in the purification and characterisation of pairing activities from mammalian cells.
The objective of the present study was to isolate and characterise a pairing activity from, a meiotic tissue of a mammalian system - the rat testis. Meiosis constitutes one of the important phases of the process of spermatogenesis. In rat, the various stages of spermatogenesis (the meiotic stages and spermiogenesis) can be broadly demarcated by the age of the rat.
The pachytene stage of meiosis in rat is spread over nine days. Rat testicular nuclear extracts from animals, aged 38-42 days (the stages of spermiogenesis were absent and a significant proportion of the cells were in the pachytene stage during this period), were fractionated sequentially on phosphocellulose, heparin-agarose and ssDNA-cellulose columns. Fractions were tested for the ssDNA-dependent formation of slow-moving products on an agarose gel, using the strand-transfer away with M13mp19 RF III and ssDNA substrates. A partially purified fraction, which catalysed the formation of such products, was obtained from the ssDNA-cellulose column on eluting with 0.6 M KCI.
In order to characterise the products formed by this partially purified fraction, they were analysed by electron microscopy. The majority of the observed structures represented multimers of the linear substrate. A significant proportion of the products resembled forked (Y-branched) structures and paired (duplex-duplex paired and 6s-duplex paired) structures. Length measurement of the multimers indicated the presence of a ligase activity in the partially purified fraction. The arms of the Y-branched structures were equal in length. The length of the molecule from the end of the stem to the end of either of the arms was the same as that of a monomer. The Y-branched structures were duplex in nature along their entire length. Exonucleases were not detected in the fraction; hence, these structures might actually represent paired structures. Typical r- and &-structures were not observed.
The EM studies indicated that the slow-moving products, observed on the gel, might be ligated structures. The mobility of these products were found to be similar to those formed by a standard ligase - the T4 DNA ligase. Restriction digestion of the junctions of the ligated products revealed that the ends were intact and there was no nucleotide loss, as seen in the case of ligases involved in nonhomologous recombination. In order to characterise the type of ligase present, radiolabelled a-ATP was used to form the ligase-AMP adduct which was then separated by SDS-PAGE. Autoradiography revealed the presence of a major 100 kDa polypeptide (most probably DNA ligase I or 111), with a minor 65 kDa polypeptide (most probably DNA ligase 11). The latter, in turn, was found to be enriched in the ssDNA-cellulose fraction eluting with 0.8 M KC1. The formation of the ligated products was strictly dependent on the presence of ssDNA. Other characteristics of the ligase activity included the substitution of ATP by UTP. The ligase present in the 0.8 M fraction was found to be inhibited by ssDNA.
Experiments to test the heat - stability of the products revealed the presence of a ssDNA-aggregating activity in the partially purified fraction. This aggregation was found to be specific for linear ssDNA. In the strand-transfer assay, the first step involves the transfer of the ssDNA circle on the linear duplex substrate accompanied by the partial displacement of the non-complementary strand of the duplex. It was reasoned that the linear end generated (partially-displaced strand) would be aggregated by the linear ssDNA-aggregating activity, thereby preventing the progression of the reaction and accounting for the absence of V - and a-structures under the EM. The formation of such aggregates was therefore monitored on the gel, using radiolabelled linear dsDNA. The formation of theme aggregates was found to be dependent on ssDNA and homology. ATP was essential for the reaction, though ATP hydrolysis was not necessary, since ATPW could substitute for ATP in the reaction. This suggested the presence of a pairing activity in the partially purified fraction.
The aggregating activity was found to be enriched in the 0.5M KCl-fraction from the ssDNA-cellulose column. The molecular weight of the enriched polypeptide was found to be similar to that of histone Hl. Western blot analysis of the polypeptide with monospecific antibodies against histone Hl a and a comparison of the protein fingerprinting pattern of the polypeptide with that of histone Hla revealed the identity of the polypeptide to be histone Hla. Formation of stable protein-free aggregates of linear ssDNA was found to be a common property of histone H1 subtypes. This aggregation of linear ssDNA (non-complementary) did' not require ATP or Mg2+. Purified hietone H1 subtypes, however, could not cause homology-dependent aggregation of the linear duplex substrate.
This study thus indicates the presence of a pairing activity and a ligase activity in a partially purified fraction from rat testicular nuclear extracts. Unlike other eukaryotic pairing activities characterised so far, this preparation is free from exonucleases. The homology-dependent duplex DNA aggregates, observed in this study, may therefore represent intermediates actually generated by the pairing activity.
School:Indian Institute of Science
Source Type:Master's Thesis
Keywords:zoology rat dna testis
Date of Publication:07/01/1994