Biochemistry 401G Lecture 33/34 Andres DNA Replication: DNA replication is the most fundamental and critical part of chromosome replication, which takes place only once during each cell division. It results in the exact duplication of the double stranded DNA helix, yielding two identical copies of the original. The reactions we are about to discuss differ from reactions in intermediate metabolism in that they require the direct involvement, and ultimately are controlled by, genetic information (in the form of template DNA/RNA). We will see that the template plays a passive role, determining which specific substrates are bound, whereas the enzyme continues to specify the nature of the chemical reaction once substrate is bound. Semiconservative Replication: Watson and Crick during the formulation of their model for the DNA double-helix proposed that during replication one of the strands in each DNA molecule is newly synthesized (new strand or Daughter), whereas the other is an unaltered strand from the parental DNA molecule (template strand). This is termed "Semiconservative." Which features of the Watson and Crick model led to this hypothesis? 1) The bases are capable of hydrogen bonding capability 2) This hydrogen bond directed base pairing (AT and GC) determines the specificity of the interaction between the two strands of DNA which constitute the double helix. Messelson and Stahl In 1957, Matthew Meselson and Franklin Stahl did an experiment to determine which of the following models best represented DNA replication: • Conservative: Two parental strands stay together, and two daughter

strands stay together. • Dispersive: Parental and daughter material is mixed on each strand. • Semiconservative

While replication is conceptually simple, it is mechanistically complex.

Several general features of DNA replication: 1) Semiconservative. 2) Ordered and Sequential. Begins at fixed points and occurs simultaneously with parental DNA unwinding. 3) Replication uses activated substrates (dNTP's). 4) DNA replication is discontinuous. The need to extend DNA chains of opposite polarity within the same replication fork means one chain grows in the opposite direction of the other. 5) Replication is far more accurate than any other enzyme-catalyzed process.

DNA replication depends upon DNA polymerase: DNA Polymerase: Catalyzes the creation of a phosphodiester bond between nucleotides in a DNA chain. The 3' hydroxyl group at the end of a DNA polymer reacts with the 5' alpha phosphate of an incoming dNTP. The dNTP has been positioned into the growing (primer strand) by H-bonding with the appropriate base on the template strand.

DNA polymerase only adds dNTP's to the 3' end of an existing DNA chain because each successive nucleotide residue is added to the 3' end of the nucleic acid. RESULT: chain growth is always 5' to 3' direction. This is termed the 5' to 3' polymerase reaction. The DNA template can be single or double stranded but is essential. A primer chain with a free 3' hydroxyl is also required. These enzymatic properties mean that the DNA polmerization reaction is unidirectional (5' to 3') and requires both template, primer, and dNTPs.

There are two important bacterial polymerases I and III. General DNA polymerase characteristics: 1. Polymerase will only elongate existing polynucleotides. It cannot initiate polynucleotide formation. 2. Will only catalyze 5' to 3' synthesis. 3. DNA polymerase is unable to unwind duplex DNA to separate the two

strands, which need to be copied. Why do DNA/RNA polymerases always catalyze 5’ to 3’ synthesis?

DNA replication in E. coli Some of the problems to overcome during DNA synthesis: Where to start? Separation of the two DNA strands. Keep them apart. Make a primer. Make DNA-this alone requires many proteins. Identification of a replication origin: Initiation The E. coli genome is circular duplex DNA and contains a single origin of replication. DNA duplication in bacteria begins at a specific site in the DNA called "oriC". The melting of the DNA duplex at this point is used to initiate DNA replication and requires the acrtion of several proteins. The dnaA gene product (dnaA protein): ATP is needed for dnaA protein to bind to the OriC repeats. This binding results in the opening of the DNA duplex at the oriC site. DnaA protein is therefore necessary for the initiation of replication.

The dnaB gene product (dnaB protein): Helicase and ssb protein (single-stranded binding protein). DnaB is a helicase. A helicase moves along the DNA strand and opens the duplex by separating the DNA strands. The binding of dnaB near the oriC DNA region requires ATP. Two helicases bind to the oriC region, one helicase for each strand of the DNA. The separated strands are prevented from reannealing by the binding of ssb protein. The dnaG gene product: Primase. The primase catalyzes the synthesis of short RNA molecules that function as primers for DNA synthesis by E. coli DNA polymerase III (pol III). The primase binds to dnaB and in this complex (primosome) provides RNA primers (9-12 nucleotides in length) for both strands of the duplex DNA.

Synthesis of DNA: Elongation After the synthesis of a short 9-12 nucleotide RNA primer, DNA pol III holoenzyme enters the replication fork and is able to utilize the RNA as a primer for DNA synthesis. As the replication fork opens up, the leading strand synthesis can continue (because of the 5' to 3' direction of synthesis), but a gap develops in the lagging strand. The leading strand: Synthesis continues in an uncomplicated way, 5' to 3' as the helicase unwinds the parental DNA double strand. The resulting double stranded DNA product is antiparallel.

DNA pol III is a large multiprotein enzyme complex (holoenzyme) which is somewhat dimeric in nature (there are two active sites in the complex). Primase can bind to the pol III complex, but the arrangement of the DNA strand as it passes through the Pol III/primase complex is quite unique. It forms a loop structure such that primase and pol III active site can accomplish discontinuous synthesis of the lagging template strand even though the general direction of the pol III complex is opposite to the required direction of DNA synthesis.

After primase makes another primer on the lagging template, the adjacent Pol III active site can extend the primer (incorporating dNTP's) by utilizing the same loop structure and feeding the template past the active site. However, the lagging strand loop cannot be feed through the Pol III complex forever, and after a nascent DNA strand is synthesized the loop is released and a new one is formed using the open DNA further up the fork.

This process results in the production of a series of short pieces of DNA, each with an RNA primer at the 5' end. These are called OKAZAKI FRAGMENTS. Once the DNA polymerase reaches the 5' end of the previous Okazaki fragment three events must occur: 1) Removal of the RNA PRIMER to produce a gap. 2) Replacement of the gap with DNA. 3) Covalent joining of the NICK left in the lagging strand.

Two additional enzymes are needed for the completion of these actions. DNA Polymerase I: In bacteria this enzyme has an added enzymatic function: a 5' to 3’ exonuclease (catalyzes hydrolysis of phosphodiester bonds at the ends of nucleic acid molecules). These activities allow the enzyme to carry out a NICK TRANSLATION REACTION- removal of ribonucleotides from the 5' end of the RNA primer, coupled with simultaneous extension of the DNA strand from the 3' end of the Okazaki fragment. The NICK is the single strand interruption of the duplex DNA, in which no nucleotides are missing. A GAP is similar but one or more nucleotides are missing. Once the ribonucleotides have been removed the DNA polymerase I can not close the remaining NICK.

DNA LIGASE catalyzes the formation of a phosphodiester bond between these ends.

In Eukaryotes the process of DNA replication seems to be similar. Synthesis of bulk DNA: Eukaryotes use two different polymerases in place of Pol III 1. DNA polymerase delta is used to synthesize the leading strand (highly processive). 2. DNA polymerase alpha synthesizes the lagging strand. 3. There is a ubiquitous DNA polymerase epsilon that may be essential.

Linear Genomes: Special problem arises in completing the lagging strand, excision of the RNA primer leaves a gap that can not be completed by DNA polymerase. Since polymerase adds bases in the 5' to 3' direction only, we can not get complete duplication of the 5' end of the daughter strand. Therefore, with each round of replication we would lose information at the end of the chromosomes (i.e. the telomeres). If this were to occur, the chromosome would shorten a bit during each cell division.

In eukaryotes this problem is solved by the addition of TELOMERES at the ends of each chromosome. Telomeric DNA consists of simple tandemly repeated sequences (Human: repeat TTAGGG). These sequences are repeatedly added to the 3' termini of chromosomal DNA's (often hundreds of copies) by enzymes called telomerases. This elongation allows room for a primer to bind and initiate synthesis on the other strand maintaining the approximate length of the chromosome and preventing the loss of coding sequence. NOTE: the telomerase must function without a primer.

Telomerase can elongate the lagging strand template from its 3' hydroxyl end (i.e. in a 5' to 3' direction). Telomerase- Is a polymerase (a reverse transcriptase) that carries a piece of RNA in its active site (to serve as a template). This RNA hybridizes to the repetitive sequence at the telomeric end of the lagging strand template and synthesizes additional telomer repeats to this strand.

Fidelity of DNA Replication: DNA replication is very fast: In bacteria replisomes travel at 1000bp per second. This means that the polymerase has 1 millisecond to verify the correct nucleotide has been incorporated. However, the process is very accurate: less than 1 error occurs in every 10 million nucleotides incorporated into the growing DNA chain. DNA replication is therefore by far the most accurate of known enzyme catalyzed processes: for humans about 10-6 mutations per generation occur. This means that about 6 errors are made in your DNA (3 x 109 bp) each time a cell divides (due to the accuracy of the polymerase and the action of additional repair systems). This accuracy can not be explained by H-bonding which would result in an error rate of 0.1% to 1.0%.

DNA polymerase has a 3' to 5’ exonuclease activity that allows it to recognize and remove mismatched nucleotides. There are additional repair systems that we will discuss in later classes.

Replication of Viral Genomes (AIDS Therapy): Many virus including the Retroviruses that are responsible for many tumors and AIDS contain RNA genomes. How are they replicated? Most RNA viruses contain a genome that consists of a single molecule of single-stranded RNA. For the retroviruses the RNA genome achieves latency- the ability to persist in the host for a long period of time without causing disease- by making a DNA copy of the genome and inserting the copy into the host genome. The enzyme Reverse Transcriptase, an enzyme that enters the cell along with the RNA genome, makes this copy. The reverse transcriptase lacks proof reading activity and therefore the virus undergoes a high rate of mutagenesis. This results in the generation of many virus copies that are unable to survive but also a greatly accelerated rate of evolution. This process helps the virus evade host defense systems. The key role of reverse transcriptase during the retroviral lifecycle has led to the isolation of specific inhibitors for the enzyme. One such inhibitor, AZT is used as an anti-AIDS drug. This nucleoside is taken into cells and phosphorylated. In this state AZT can be incorporated into DNA where it acts as a chain terminator much like dideoxyribonucleotides (we will discuss these compounds again when we see to sequence DNA). Most DNA polymerases have a very low affinity for AZT, but HIV reverse transcriptase binds the drug very effectively. Thus, AZT triphosphate is an effective competitor for dTTP in HIV-infected cells.