|
DnaG is a bacterial DNA primase and is encoded by the ''dnaG'' gene. The enzyme DnaG, and any other DNA primase, synthesizes short strands of RNA known as oligonucleotides during DNA replication. These oligonucleotides are known as primers because they act as a starting point for DNA synthesis. DnaG catalyzes the synthesis of oligonucleotides that are 10 to 60 nucleotides (the fundamental unit of DNA and RNA) long, however most of the oligonucleotides synthesized are 11 nucleotides.〔 These RNA oligonucleotides serve as primers, or starting points, for DNA synthesis by bacterial DNA polymerase III (Pol III). DnaG is important in bacterial DNA replication because DNA polymerase cannot initiate the synthesis of a DNA strand, but can only add nucleotides to a preexisting strand. DnaG synthesizes a single RNA primer at the origin of replication. This primer serves to prime leading strand DNA synthesis. For the other parental strand, the lagging strand, DnaG synthesizes an RNA primer every few kilobases (kb). These primers serve as substrates for the synthesis of Okazaki fragments. In ''E. coli'' DnaG associates through noncovalent interactions with bacterial replicative helicase DnaB to perform its primase activity, with three DnaG primase proteins associating with each DnaB helicase to form the primosome.〔 Primases tend to initiate synthesis at specific three nucleotide sequences on single-stranded DNA (ssDNA) templates and for ''E. coli'' DnaG the sequence is 5'-CTG-3'.〔 DnaG contains three separate protein domains: a zinc binding domain, an RNA polymerase domain, and a DnaB helicase binding domain. There are several bacteria that use the DNA primase DnaG. A few organisms that have DnaG as their DNA primase are ''Escherichia Coli'' (''E. coli''), ''Bacillus stearothermophilus'', and ''Mycobacterium tuberculosis'' (MTB). E. coli DnaG has a molecular weight of 60,000 kilodaltons (kDa) and contains 581 amino acids. ==Function== DnaG catalyzes the synthesis of oligonucleotides in five discrete steps: template binding, nucleoside triphosphate (NTP) binding, initiation, extension to form a primer, and primer transfer to DNA polymerase III. DnaG performs this catalysis near the replication fork that is formed by DnaB helicase during DNA replication. DnaG must be complexed with DnaB in order for it to catalyze the formation of the oligonucleotide primers.〔 The mechanism for primer synthesis by primases involves two NTP binding sites on the primase protein (DnaG). Prior to the binding of any NTPs to form the RNA primer, the ssDNA template sequence binds to DnaG. The ssDNA contains a three nucleotide recognition sequence that recruits NTPs based on Watson-Crick base pairing.〔 After binding DNA, DnaG must bind two NTPs in order to generate an enzyme-DNA-NTP-NTP quaternary complex. The Michaelis constant's (km) for the NTPs vary depending on the primase and templates. The two NTP binding sites on DnaG are referred to as the initiation site and elongation site. The initiation site is the site at which the NTP to be incorporated at the 5' end of the primer binds. The elongation site binds the NTP that is added to the 3' end of the primer. Once two nucleotides are bound to the primase, DnaG catalyzes the formation of a dinucleotide by forming a phosphodiester bond via dehydration synthesis between the 3' hydroxyl of the nucleotide in the initiation site and the α-phosphate of the nucleotide in the elongation site. This reaction results in a dinucleotide and breaking of the bond between the α and β phosphorus, releasing pyrophosphate. This reaction is irreversible because the pyrophosphate that is formed is hydrolyzed into two inorganic phosphate molecules by the enzyme inorganic pyrophosphatase. This dinucleotide synthesis reaction is the same reaction as any other enzyme that catalyzes the formation of DNA or RNA (DNA Polymerase, RNA Polymerase), therefore DnaG must always synthesize oligonucleotides in the 5' to 3' direction. In ''E. coli'', primers begin with a triphosphate adenine-guanine (pppAG) dinucleotide at the 5' end. In order for further elongation of the dinucleotide to occur, oligonucleotide must be moved so that the 3' NTP is transferred from the elongation site to the initiation site, allowing for another NTP to bind to the elongation site and attach to the 3' hydroxyl of the oligonucleotide. Once an oligonucleotide of appropriate length has been synthesized from the elongation step of primer synthesis, DnaG transfers the newly synthesized primer to DNA polymerase III for it to synthesize the DNA leading strand or Okazaki fragments for the lagging strand.〔 The rate limiting step of the primer synthesis occurs after NTP binding but before or during dinucleotide synthesis.〔 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「DnaG」の詳細全文を読む スポンサード リンク
|