Molecular Biology: General Theory

Transcription

1 Molecular Biology: General Theory Author: Dr Darshana Morar Licensed under a Creative Commons Attribution license. DNA REPLICATION DNA replication is the process of duplicating the DNA sequence in the parent strand to produce an exact replica (daughter strand). Replication is semi-conservative: each one of the two parental strands serves as a template for the new strand synthesis; therefore, duplicated double helices contain one parental strand and one daughter strand. DNA polymerases are the enzymes responsible for DNA synthesis. These enzymes use a single-stranded DNA template to catalyze the polymerization of a complementary DNA strand. In a cell, DNA replication must happen before cell division can occur. DNA synthesis begins at specific locations in the genome, called "origins", where the two strands of DNA are separated. RNA primers attach to single stranded DNA and the enzyme DNA polymerase extends the primers to form new strands of DNA, adding nucleotides matched to the template strand. The unwinding of DNA and synthesis of new strands forms a replication fork. In addition to DNA polymerase, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA replication can also be performed artificially, using the same enzymes used within the cell. DNA polymerases and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs artificial synthesis in a cyclic manner to rapidly and specifically amplify a target DNA fragment from a pool of DNA. The replication process in bacteria The chromosome of a prokaryote is a circular molecule of DNA. Replication begins at one origin of replication and proceeds in both directions around the chromosome. The basic steps of DNA replication are: Initiation replication begins at an origin of replication Elongation new strands of DNA are synthesized by DNA polymerase 1 P age

2 Termination replication is terminated differently in prokaryotes and eukaryotes Initiation This step involves the assembly of a replication fork (bubble) at an origin of replication sequence of DNA found at a specific site of the circular chromosome of a bacterium. This origin of replication is unwound, and the partially unwound strands form a "replication bubble", with one replication fork on either end. Each group of enzymes at the replication fork moves away from the origin, unwinding and replicating the original DNA strands as they proceed. The factors involved are collectively called the pre-replication complex. It consists of the following: A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins (SSB) swiftly bind to the separated DNA, thus preventing the strands from reuniting. Because DNA is helical, this unwinding means that the DNA needs to rotate to avoid too much supercoiling. An enzyme, named DNA topoisomerase I, precedes the replication complex, and cleaves one strand of DNA ahead of the replication machinery, allowing free rotation of the DNA between the nick and the replication complex. A primase (an RNA polymerase), which generates an RNA primer that serves as a starting point or primer for synthesis of the new DNA chain. A DNA polymerase III holoenzyme, which in reality is a complex of enzymes that together perform the actual replication. Elongation After the helicase unwinds the DNA, single-strand binding protein is used to hold the DNA strands apart. RNA primase is then bound to the starting DNA site, and it synthesizes an RNA primer complementary to the DNA. At the beginning of replication, an enzyme called DNA polymerase III holoenzyme binds to the RNA primer, which indicates the starting point for the replication. DNA polymerase can only synthesize new DNA from the 5 to 3 (of the new DNA - i.e. it moves on a template from 3 to 5 ). Because of this, the DNA polymerase can only travel on one of the original DNA strands without any interruption. This original strand, which goes from 3 to 5, is called the leading strand (Fig. 17). The complement of the leading strand, from 5 to 3, is the lagging strand. The holoenzyme catalyses the formation of a phosphodiester bond between the 3 -OH group of the last sugar on a chain of DNA (the primer), and the 5 phosphate group on an incoming nucleotide triphosphate, chosen for its complementarity to the facing nucleotide present on the template strand (the one being copied). 2 P age

3 Figure 6 The replication fork Replication of the lagging strand is more complicated than that of the leading strand. The orientation of the lagging strand is opposite to the working orientation of DNA polymerase III (which can only synthesize new DNA from the 5 to 3 ). As a result, the DNA of the lagging strand is replicated in a piecemeal fashion. The primase, which accompanies the holoenzyme, synthesizes RNA primers along the lagging strand every few hundreds of base pairs, which are then used as primers for DNA polymerase III action. Small stretches of DNA (so-called Okazaki fragments, ~2 kb), are synthesized until the next RNA primer which prevents DNA polymerase III activity. Then DNA polymerase I enters into action, digesting the RNA primer, and replacing ribonucleotides with deoxyribonucleotides. Finally DNA ligase generates the last phosphodiester bond between two newly synthesized Okazaki fragments. Figure 7 DNA replication in bacteria (Taken from: ) 3 P age

4 DNA Helicase is an enzyme that unravels the DNA double helix and breaks the hydrogen bonds. DNA primase is an enzyme that generates an RNA sequence that serves as a starting point or primer for synthesis of the new DNA chain. DNA polymerases are enzymes that synthesize a complementary DNA strand using the original strand as a template. In DNA synthesis, the new strand grows 5' to 3'. An Okazaki fragment is a stretch of non-parental DNA produced along the lagging strand of parental DNA by the DNA polymerase beginning at primer. Three DNA polymerases are found in bacteria 1) DNA polymerase I (Pol I) functioning essentially in DNA repair, and a little in DNA replication. DNA polymerase I possesses three enzymatic activities: 5' to 3' (forward) DNA polymerase activity, requiring a 3' primer site and a template strand. DNA polymerase I catalyzes the addition of nucleotides to the 3 hydroxyl of primer DNA and requires dntps and Mg 2+. Synthesis is always in the 5 to3 direction. A 5 to 3 (forward) exonuclease activity that mediates nick translation during DNA repair. A 3 to 5 (reverse) exonuclease activity that allows proofreading and mediates removal of RNA primers during replication. 2) DNA polymerase II (Pol II) is a DNA repair enzyme involved in replication of damaged DNA. It has 3' to 5' exonuclease activity that mediates proofreading. DNA polymerase II differs from DNA polymerase I in that it lacks 5' to 3' exonuclease activity and cannot use a nicked duplex template. 3) DNA polymerase III (Pol III) is the main enzyme used in DNA replication; it is a complex of several proteins (at least 20), forming the so-called holoenzyme. DNA polymerase III has 5' to 3' (forward) DNA polymerization activity and catalyzes the addition of dntps to the end of a new DNA strand with release of inorganic pyrophosphate (PPi). It also has 3' to 5' exonuclease proofreading capability. The holoenzyme has high processivity, i.e. the number of nucleotides added per binding event. The rate of DNA synthesis is extremely fast: 30-60,000 nucleotides per minute. 4 P age

5 Termination Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. Termination of DNA replication in E. coli is regulated through the use of termination sequences (Ter sites) and the Tus protein (terminator utilization substance protein). The Tus protein binds to the termination sites, these Tus -Ter complexes allow a replication fork to pass through in one direction, but not the other. Multiple Ter sites are located in the termination region of the E. coli chromosome and slow down and stop the movement of the replication forks in this region. As a result, the replication forks are constrained to always meet and terminate within the termination region of the chromosome. Fidelity of DNA replication The error rate in DNA replication is very low and has been estimated at 1 error in 10 million bases. DNA polymerase III proofreads the newly made strand before continuing with replication. When an incorrect nucleotide is incorporated, the 3 end will be frayed. The DNA polymerase III recognizes the problem, "backs up" and removes the incorrect nucleotide by means of its 3' to 5' exonuclease activity. The correct nucleotide is then added to the chain and elongation is resumed. Note: DNA polymerase III present in the replication fork has three important properties: (i) Chain elongation (ii) Processivity and (iii) Proofreading Catalytic activities of DNA polymerases: Important note: DNA polymerases I, II and III carry both a 5 to 3 polymerase activity, and a 3 to 5 exonuclease activity which eliminates from an elongating chain of DNA any misincorporated (non complementary) nucleotides. This is known as proofreading activity. 5' to 3' polymerase activity for DNA synthesis (DNA Pol I, II and III) 5' to 3' exonuclease activity for removal of the DNA/RNA primer (allows DNA Pol I to destroy a strand of DNA or RNA located ahead of it (3 to it) on the DNA template). 3' to 5' exonuclease activity for proofreading (DNA Pol I, II and III) The replication process in viruses Viral populations do not grow through cell division, because they are acellular; instead, they use the machinery and metabolism of a host cell to produce multiple copies of themselves. In many ways the 5 P age

6 replication is very similar to that of bacteria but the expression of virus genetic information is dependent on the structure of the genome of the particular virus concerned. In every case, the genome must be recognized and expressed using the mechanisms of the host cell. The replication process in Eukaryotes The DNA replication process in eukaryotes is essentially the same as in prokaryotes, with some differences. While replication begins at ori C in prokaryotes, there are multiple origins of replication in eukaryotes due to the sheer size of the chromosomes. Replication begins at specific places on the chromosome - the origin or "ori" region and proceeds bidirectionally. As in prokaryotes, the two parent strands are unwound with the help of DNA helicases. Stabilizing proteins attach to the unwound strands, preventing them from winding back together. DNA polymerases α and δ are responsible for DNA synthesis in eukaryotes. DNA polymerase α begins with an RNA primer and then adds a few DNA nucleotides. DNA polymerase δ takes over and synthesizes DNA nucleotides at approximately 100 times the rate of DNA polymerase α. RNA primers, needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments, are synthesized by subunits of DNA polymerase α. DNA Polymerases β and ξ are presumed to be involved in DNA repair in eukaryotes which may indicate that they are involved in RNA primer removal. Finally, as in prokaryotes, each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by DNA ligase. Eukaryotic nuclear chromosomes are packaged by histone proteins into a condensed structure called chromatin. The replication of eukaryotic chromosomes presents additional problems, including the need to remove, replicate and replace histones and other similar proteins associated with the DNA double helix. In addition, eukaryotes must be able to deal with the linear ends of each chromosome arm (the telomeres), i.e. once the RNA primer is removed at the end of each arm of a chromosome, there is no 3 - OH end for the DNA polymerase to recognize and use to replace missing nucleotides. This function is accomplished by telomerase, an enzyme that adds specific DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3 end of DNA strands in the telomere regions. This enzyme is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres. 6 P age

7 Figure 8 The replication of DNA in Eukaryotes 7 P age