Purification and Characterization of a DNA Helicase from Pea Chloroplast that Translocates in the 3′‐to‐5′ Direction

Abstract

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2' or MnZ+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.Keywords: unwinding enzyme ; DNA helicase ; DNA-dependent adenosine triphosphatase ; chloroplast ; replication.The mechanism of DNA replication has been well defined in plasmids, bacteriophages, bacteria, viruses and, to a lesser extent, in yeast [1]. Most studies of the replication of DNA in plants have investigated chloroplast DNA [2]. The replicative intermediates of chloroplast DNA molecules of 120-160 kbp from higher plants are well characterized and the origins of replication have been mapped for pea chloroplast DNA and Chlumydonzonus chloroplast DNA [3-61. Of the enzymes involved in DNA replication, only DNA polymerase [7] and DNA topoisomerase [8, 91 have been purified from chloroplasts. The presence of DNA primase has also been reported in pea chloroplasts (101. DNA helicases are an important class of enzymes that are involved in replication, recombination and repair of DNA [I, 11, 121. These enzymes unwind duplex DNA and provide single-stranded DNA template. Many DNA helicases have been isolated from bacteriophage, bacterial, viral and eukaryotic systems [ l l , 121. Multiple DNA helicases are present in a cell because of the different structural requirements of the substrates at various stages of repair, replication and recombination. In Escherichia coli functions for most of the DNA helicases have been attributed [ll]. Six DNA helicases have been isolated from human cells by . In plants, there are two reports of DNA helicases, one in lily [19] and the other in soybean [20]. In both of these studies, the enzymes were not purified to homogeneity or extensively characterized.We now report the purification and detailed characterization of a DNA helicase from pea chloroplast, which is a homodimer of 68-kDa subunits and requires divalent cations for activity.

The ArabidopsisAt1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities

“…The pea enzyme is larger (~90 kDa) than the Arabidopsis Twinkle homologue, but it was not characterized for DNA helicase activity. CtDNA replication involves multiple replication origins and bidirectional DNA synthesis [42,43], which would require DNA primase activity for lagging strand synthesis.…”

A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase

“…The chloroplast lysate was prepared as described (Tuteja et al 1996). The MCM6 protein from cytosol, nuclei, chloroplast or overexpressed bacteria and transgenic plant was resolved on SDS-PAGE.…”

“…This partial duplex DNA substrate contained hanging tails of 15 nucleotides at both the 5 0 and 3 0 ends. It was prepared as described earlier (Tuteja et al 1996). Other forked or nonforked substrates and direction specific substrates were also prepared as described earlier (Tuteja et al 1994;Tuteja et al 1996).…”

“…It was prepared as described earlier (Tuteja et al 1996). Other forked or nonforked substrates and direction specific substrates were also prepared as described earlier (Tuteja et al 1994;Tuteja et al 1996). The DNA helicase reaction was performed for 60 min at 37°C using the purified MCM6 protein (250 ng) and the reaction was analyzed as previously described (Tuteja et al 1994).…”

“…The ATPase reaction conditions were identical, except that the 32 P-labeled helicase substrate and 2 mM ATP were omitted and instead 1665 Bq (c-32 P)-ATP was included. This reaction was run for 2 h at 37°C and analyzed as described (Tuteja et al 1994(Tuteja et al , 1996.…”