DNApol then (4) adds deoxyribonucleotide triphosphates to the 3'-end of the primer until the molecules are completely replicated.

Transcription

1 Biochemistry II DNA Synthesis p-p-p-n-oh ssna is a nucleotide (of any particular base, as represented by the N) with a 5' triphosphate and a 3' hydroxide. This module is on DNA so the nucleotide is dntp unless otherwise noted. Single stranded nucleic acid. I. Producing DNA, enerally The production of DNA is called many things, but most commonly: DNA Replication DNA Synthesis It is the process by which DNA molecules are made. The enzyme responsible for making DNA is called DNA polymerase (DNApol). It makes DNA, by joining nucleotides, at a rate ranging from about 50 to over 1000 nucleotides per second (depending upon the organism). The substrate for the enzyme is dsdna, dntp, and an incomplete single, complimentary strand base paired to a segment of the dsdna. The reaction is: (DNA) n + dntp -> (DNA) n+1 + PPi So elongating a DNA chain would look like: 5'-p-ATTA-OH-3' + p-p-p-n-oh -> 5'-p-ATTAN-OH-3' + pp The region of a DNA molecule where replication starts is called the origin of replication. The origin is a certain sequence. DNApol (1) finds the origin of replication on a dsdna molecule, and (2) pries the chains apart at that spot. The chains are barred with another protein that only binds single strands then (3) short pieces of RNA (primer) are produced on both strands of the pried open region. 1 AAATTTAAATTTAAATTTAAATTTAAATTTAAATTT AAAAAAAAA i TTTAAATTTAAATTTAAA 2 AAATTTAAA TTTAAATTT AAATTTAAA AAAAAATTT TTTAAATTTAAATTTAAA aaagg 3 AAATTTAAA TTTAAATTT ccctt AAAAAATTT AAATTTAAA DNApol then (4) adds deoxyribonucleotide triphosphates to the 3'-end of the primer until the molecules are completely replicated. TTTAAATTTAAATTTAAA aaaggttt T 4 AAATTTAAA TTAAATTT AATTTAAA ccctttaaa A AAAAAATTT Note the word replicated. DNA molecules are NOT made from scratch; they are copies of existing DNA molecules! DNApol produces copies! The strands that are used to make the copies are called templates. All polymerase enzymes reads their templates from 3' to 5'. The region where synthesis is going on is called the replication fork.

2 II. Prokaryote Replication Prokaryotic replication starts when replication initiation protein, dnaa, binds to the origin of replication and starts prying the chains apart at that site. As the chains open up, single strand regions are exposed and helicase enzymes bind and open the chains wider allowing access to single strand binding protein, SSB, (which hold the chains open). In prokaryotes, there are three primary polymerase enzymes: pol I pol II pol III For finishing replication, repair, and recombination For repair and restarting stalled replication The main replication enzyme Primase enzyme then produces a short stretch of RNA (primer) on the template strands before pol III comes in and starts adding nucleotides to the primer. Now, polymerase enzymes can only synthesize in the 3' to 5' direction. This presents a problem because there is a 3' to 5' region of the fork and a 5' to 3' region at the fork; how does an enzyme that only does 3' to 5' replicate on each strand? It can t read this in the correct <<< direction 5'-TTTAAATTTAAA aaagg TTTAAATTT-3' AAATTTAAA-5' ccctt 3'-AAA It can read this >>> in the correct direction The answer is by looping the 5' to 3' strand... The looped strand is called the lagging strand. The strand that s not looped is called the leading strand.

3 By looping the lagging strand, polymerase at the replication fork can read both strands in the same direction; however, not indefinitely. The lagging strand 3' to 5' region is much shorter, so eventually, polymerase will need to let go and re-loop that strand. Hence the name, lagging. Replication is not as fast on that strand because it must constantly be readjusted. It lags behind! Because the lagging strand must be looped, then re-looped, it also means that replication must be restarted each time the strand is re-looped. otta make a new primer and start chain elongation again. onsequently, instead of one long chain on the lagging strand, you end up with a lot of fragments. These fragments are called Okazaki fragments. X

4 The elongation of the DNA chains is not totally sequential, but only slightly processive, occurring ~20 nucleotides at a time. Other activities are found on the polymerase enzyme. These activities are EXONULEASES. Exonucleases are degradatory activities which remove nucleotides or very short stretches of sequence from the 5' or 3' end of a nucleic acid chain. Note that these are different from ENDONULEASES, which act by cleaving some internal area of the nucleic acid chain. PROOF READIN OMPONENT: In addition to the chain elongation component of the enzyme, there is also a proof-reading component. This is a 3'->5' EXONULEASE that reacts as shown; This component removes bases from the 3' end of the growing chain if they are improperly paired with the template. The elongation of the chain cannot proceed unless U,the incorrectly paired base is removed. The activity has the effect of ensuring that the errors found in a DNA chain is kept to a minimum. ERROR ORRETIN ATIVITY: Another key component for insuring the accuracy of replication is a 5'->3' EXONULEASE. This activity is enhanced by concomittent DNA synthesis and has an active site that is distinct from the 3'->5' exonuclease and the polymerase activity. Its primary function is to remove pyrimidine dimers and the RNA primer, which is required for the initiation of the elongation reaction. Now, all of these activities make up the polymerase HOLOENZYME. The structure of the enzyme is shown below. 5'->3' exo 3'->5' exo polymerase - term small fragment large fragment or Klenow Oddly, these large and small fragments may be divided into active regions by cleavage of the enzyme with a PROTEASE. The Klenow fragment has a large and small domain (the polymerase residing in the large domain and the 3'->5' exonuclease in the smaller). Polymerization proceeds in the cleft of the LARE domain and as shown in the scheme below. Two other polymerases found in cells are DNA pol III, and DNA pol II. Pol III resembles the KLENOW and mostly functions in the initiation of the polymerization reaction (pol I erases primer and fills in the gaps). The function of pol II is unknown.

5 III. The replication reaction The replication reaction is coupled to denaturation of specific areas of a DNA chain, and the replication intermediate of a circular plasmid is called a THETA structure. The start site of replication may be identified by the following experiment. First, pick to distal genes. The frequency of occurrence of these genes in rapidly growing cells (or tissue) will tell you their distance from the origin of replication. Bi-directionality may be proven by: