Biochem Ch 10

DNA gyrase

(class II topoisomerase) catalyzes reaction involving relaxed circular DNA: -creates a nick in relaxed circular DNA -a slight unwinding at the point of the nick introduces supercoiling -the nick is resealed • The energy required for this process is supplied by the hydrolysis of ATP to ADP and Pi

Mismatch repair in prokaryotes

1. Mismatch in newly synthesized DNA. The correct strand is tagged with a methyl group opposite the mutation. 2. MutH, MutS, and MutL link the mismatch with the nearest methylation site which identifies the parental (correct) strand 3. An exonuclease removes the sequence of DNA from the mutated strand between proteins 4. DNA polymerase I replace the removed DNA with the correct sequence. Ligase seals the nick

Requirements for Functioning of DNA Polymerase (3)

1. all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP 2. Mg2+ 3. an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication

Properties of DNA polymerases (using E. coli as an example)

5 types total Pol I : coded by polA, exonuclease function for 5'-3' and 3'-5' Pol II : coded by polB, exonuclease function fro 3' - 5' only Pol III : coded by polC, exonuclease function fro 3' - 5' only *exonuclease = proofreading and cleaving out incorrect sequences

Telomers

A telomere is a region of repetitive noncoding nucleotide sequences at each 5' end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates, the sequence of nucleotides in telomeres is TTAGGG. Telomere shortening in humans can induce replicative senescence, which blocks cell division. This mechanism appears to prevent genomic instability and development of cancer in human aged cells by limiting the number of cell divisions

DNA polymerase III

Adding bases to the new DNA chain; proofreading the chain for mistakes

Eukaryotic DNA polymerase

At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively) Named α β γ δ ε polymerase δ is essential for replication - the principal DNA polymerase

Semi-discontinuous model for DNA replication

Because DNA polymerases only polymerize nucleotides 5' to 3', both strands must be synthesized in the 5' to 3' direction. As the helix unwinds, the other parental strand (the 5' -> 3' strand) is copied in a discontinuous fashion through synthesis of a series of fragments 1000 to 2000 nucleotides in length called Okazaki fragments -the strand constructed from Okazaki fragments is called the lagging strand -Okazaki fragments are covalently joined by DNA ligase to form an uninterrupted DNA strand Because both strands are synthesized in concert by a dimeric DNA polymerase situated at the replication fork, the 5' -> 3' parental strand must wrap around in trombone fashion so that the unit of the dimeric DNA polymerase replicating it can move in the 3' -> 5' direction -this parental strand is copied in a discontinuous fashion bc the DNA polymerase must occasionally dissociate from this strand and rejoin it further along

Cut and patch

Catalyzed by Polymerase I Cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides

Direction of replication

DNA double helix unwinds at a specific point called an origin of replication ( OriC) Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional in most organisms At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed There is one origin of replication and two replication forks in the circular DNA of prokaryotes In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin

DNA Polymerase

DNA is synthesized from its 5' -> 3' end (from the 3' -> 5' direction of the template) The 3'-OH group at the end of the growing DNA chain acts as a nucleophile. The phosphorus adjacent to the sugar is attacked, and then added to the growing chain. The leading strand is synthesized continuously in the 5' -> 3' direction toward the replication fork The lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5' -> 3' direction, but away from the replication fork lagging strand fragments are joined by the enzyme DNA ligase

3 classes of DNA polymerases

DNA-Pol I: Repair and patching of DNA. Removes RNA Primers and replaces them with DNA nucleotides DNA-Pol III: responsible for the polymerization of the newly formed DNA strand (aka adding DNA nucleotides) DNA-Pol II, IV, and V: proofreading and repair enzymes

Meselson-Stahl experiment

Determined that DNA replicates with semiconservative replication Incorporation of isotopic label as sole nitrogen source Began with parent DNA tagged with 15N First replication occured in medium with 14N --the new strands had one 15N strand and one 14N (semiconservative replication) Second replication again occured in medium with 14 N --yeilded four strands: two purely 14N and two with one 14N strand and one 15N strand (15NH4Cl). 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ultracentrifugation --so the parents are the heaviest and forms a band at the bottom of the tube, the first generation is around the middle of the tube and the purely 14N strands from the second generation are lightest at the top

UV radiation effect on DNA

Dimerization of thymine bases. Adjacent thymines bind to each other instead of binding to the adenines on the opposite strand. Creates a problem for replication or translation of that sequence. Must be cleaved out by enzymes

DNA pol-1 discovery

First DNA dependent DNA polymerase discovered Discovered by Arthur Kornberg

Evidence for semiconservative replication

Heavy DNA labeled with (15)N forms a band at the bottom of the tube, and light DNA with (14)N forms a band at the top. DNA that forms a band at an intermediate position has one heavy strand and one light strand. DNA that forms a band at an intermediate position has one heavy strand and one light strand

DNA Replication and the enzymes (7)

Making an exact duplicate of the DNA involves 30 different enzymes • Begins at an origin of replication • *Helicase* unwinds and unzips the DNA double helix • *Gyrase* (a topoisomerase) prevents the strands from tangling and cleaves DNA to help it unwind • *SSB* (single stranded binding proteins) prevents the ssDNA from re-annealing and protects it from attack by nucleases • An RNA primer is synthesized at the origin of replication by *Primase* • *DNA polymerase III* adds nucleotides in a 5′ to 3′ direction ---> can't initiate this by themselves! Need the primer • Leading strand - synthesized continuously in 5′ to 3′ direction • Lagging strand - synthesized discontinuously 5′ to 3′ in short segments; overall direction is 3′ to 5′ • *DNA polymerase I* removes the RNA primers and replaces them with DNA • When replication forks meet, *ligases* link the DNA fragments along the lagging strand • Separation of the daughter molecules is complete

Telomere synthesis

Necessary because when the primer at the 5' end of the new lagging strand is removed, there is no 3' -OH for DNA pol to attach nucleotides to, unable to fill and that sequence will be lost Adds "tails" of nucleotides to the ends of the chromosomes by binding to the DNA template with it's own RNA template, contained within the enzyme telomerase

Eukaryotic DNA Replication

Not as understood as prokaryotic. Higher level of complexity. Cell growth and division divided into phases: M, G1, S, and G2 -DNA replication takes place during the S phase Best understood model for control of eukaryotic replication is from yeast. DNA replication initiated by chromosomes that have reached the G1 phase

Oxidation Damage

Oxygen radicals, in the presence of metal ions such as Fe21, can destroy sugar rings in DNA, breaking the strand into fragments. Can be repaired by enzymes

PCNA Homotrimer

PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp Proliferating cell nuclear antigen (PCNA) is a DNA clamp that acts as a processivity factor for DNA enhances DNA synthesis elongation by associating with DNA polymerase δ hydrolyzes ATP to lock clamp around DNA; keeps DNA pol attached to template strand

Nick Translation

Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity The 5' -> 3' exonuclease activity of DNA polymerase I can remove up to 10 nucleotides in the 5' direction downstream from a 3'-OH single strand nick *If the 5' -> 3' polymerase activity fills in the gap, the net effect is nick translation by DNA polymerase

Bidirectional replication of DNA

Prokaryotes (one origin of replication) or eukaryotes (several origins) Two replication forks in the circular DNA of prokaryotes "Bubble" or "eye

Structure of DNA polymerase

Protein so it has an active site

Primase

RNA serves as a primer in DNA replication • primer activity first observed in-vivo. Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence

DNA polymerase I

Removing RNA primer, closing gaps, repairing mismatches

Eukaryotic Replication Fork

Replication is semi-conservative RNA primer formed by DNA pol α PCNA is clamped to DNA pol δ through the action of replication factor C (RFC) The enzymes FEN-1 ( 5' flap endonuclease activity) and RNase H1 degrade the RNA primers

Replication with Supercoiled DNA

Replication of supercoiled circular DNA • DNA gyrase has different role here. It introduces a nick in supercoiled DNA • a swivel point is created at the site of the nick • the gyrase opens and reseals the swivel point in advance of the replication fork • the newly synthesized DNA automatically assumes the supercoiled form because it does not have the nick at the swivel point • helicase, a helix-destabilizing protein, promotes unwinding by binding at the replication fork • single-stranded binding (SSB) protein stabilizes single-stranded regions by binding tightly to them

Prokaryotic Replication (and 2 challenges)

Separation of the two original strands and synthesis of two new daughter strands using the original strands as templates -from one 5' -> 3' strand and one 3' -> 5' strand Efficient protection from errors in replication High fidelity of replication Challenges in duplication of circular double-stranded DNA 1. Achievement of continuous unwinding and separation of the two DNA strands 2. Protection of unwound portions from attack by nucleases that attack single-stranded DNA

Base-excision repair

Sugar/phosphate backbone is present but the base is missing. Can't insert a base because DNA pol I is blocked by the backbone. A damaged base is excised from the sugar phosphate backbone by DNA glycosylase, creating an AP site (a site without a purine or pyrimidine aka apurinic or apyrimidinic). Then an apurinic/apyrimidinic endonuclease severs the DNA strand and an excision nuclease removes the AP site and several nucleotides. DNA pol I and DNA ligase then repair the gap

DNA polymerase I proofreading

The 3' → 5' exonuclease activity of DNA polymerase I removes nucleotides from the 3' end of the growing DNA chain.

Template strand

The DNA strand that provides the template for ordering the sequence of nucleotides in an mRNA transcript. Both strand of DNA are template for replication (both being copied) but only one is a template for transcription (only one mRNA being made)

Mutations, Proofreading, and repair

The removal of incorrect nucleotides immediately after they are added to the growing DNA during replication DNA replication takes place only once each generation in each cell Errors in replication (mutations) occur spontaneously only once in every *10^9 to 10^10* base pairs Can be lethal to organisms, usually not beneficial like mutations can be for viruses Proofreading - the removal (excision) of incorrect nucleotides immediately after they are added to the growing DNA during replication. Correct nucleotide added by DNA pol and sealed by ligase

Why Does DNA Contain Thymine and Not Uracil?

Thymine is more stable as compared to Uracil to mutations, thus making genetic material more stable. Stability due to the extra methyl group. Experimentally, it has been observed that RNA viruses, called retroviruses mutate more frequently than DNA viruses. This can be partly attributed to the Uracil present in RNA, which makes it more unstable.

Helicase

Unzips the DNA heli

Nucleotide-excision repair

When a serious lesion, such as a pyrimidine dimer, is detected, ABC exonuclease binds to the region and cuts out a large piece of DNA, including the lesion. DNA polymerase I and DNA ligase then resynthesize and seal the DNA. *lesions often caused by UV or chemicals

Clamp loader and sliding clamp

clamp is the term for the subunits of DNA pol III that holds core polymerase on the DNA template and move along the DNA as polymerization proceeds the clamp loader opens the sliding clamp and inserts the DNA chain

Semiconservative replication

each daughter strand contains one template strand and one newly synthesized strand

Ligase

final binding of nicks in DNA during synthesis and repair

RNA primer

short piece of RNA needed for DNA polymerase II to start provides a 3' -OH for DNA polymerase to attach the first nucleotide to --nucleotides can only be added to a 3' -OH

Gyrase

supercoiling- winds the DNA before the nick is sealed can negatively supercoil DNA ahead of replication to counteract the normal positive supercoiling and relieve torsional strain as DNA unwinds

Telomeres and cancer

telomeres are usually shortened as cells continue to divide. At some point they get so short that the cell undergoes senescence and dies. In cancer, telomerase can be found in high levels, keeping telomeres long and allowing for uncontrollable cell division to form a tumor

Synthesis and linking of new DNA strands

• begun by DNA polymerase III • the newly formed DNA is linked to the 3'-OH of the RNA primer • as the replication fork moves away, the RNA primer is removed by DNA polymerase I