DNA replication

explain how replication in eukaryotes is more complex than in prokaryotes

1) Genome size: E. coli must replicate 4.6 x 106 base pairs, whereas a human diploid cell must replicate 6 x 109 base pairs. 2)Chromosome number: E. coli is contained on 1 chromosome, whereas, in human beings, 23 pairs of chromosomes must be replicated. 3)Chromosome shape: the E. coli chromosome is circular, while human chromosomes are linear. The first two challenges are met by the use of use multiple origins of replication. In human beings, replication of the entire genome requires about 30,000 origins of replication, with each chromosome containing several hundred. -each origin of replication represents a replication unit, or replicon -the use of multiple origins of replication requires mechanisms for ensuring that each sequence is replicated only once. Proteins, called licensing factors because they permit (license) the formation of the DNA synthesis initiation complex, bind to the origin of replication. These proteins ensure that each replicon is replicated once and only once in each round of DNA synthesis. After the licensing factors have established the initiation complex, these factors are subsequently destroyed. The license expires after one use.

describe the 4 characteristics of DNA synthesis by DNA Polymerases

1) The reaction requires all 4 activated precursors: deoxynucleoside 5′-triphosphates dATP, dGTP, dCTP, TTP and the Mg2+ ion. 2) The new DNA strand is assembled directly on a preexisting DNA template. DNA polymerase is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template. 3)DNA polymerases require a primer to begin synthesis. A primer is the initial segment of a polymer that is being extended in the 5′ → 3′ direction. 4)new DNA strand grows in the 5′ → 3′ direction 5)Many DNA polymerases are able to correct mistakes in DNA by removing mismatched nucleotides. These polymerases have a distinct nuclease activity that allows them to excise incorrect bases by a separate reaction.

Replication of the E. coli Chromosome Proceeds in Stages

1)initiation 2) elongation 3) terminatiom

What has to happen to DNA before cell divides

Before each cell division our DNA must be doubled (replicated)

What are helicases

Enzymes that use the energy of ATP hydrolysis to power strand separation Usually a ring structure The helicases in DNA replication are typically oligomers containing six subunits that form a ring structure. Each subunit has a loop that extends toward the center of the ring structure and interacts with DNA. The helicase acts as a wedge to force separation of the two strands of DNA.

The Klenow fragment

First known fragments of DNA polymerase 1 Did activities 1&2

DNA polymerase 1 (from book)

Has exonuclease that removes mismatched nucleotides from 3' end of DNA by hydrolysis. The wrong nucleotide does not have the correct # of h bonds, so it flops around movement called brownian motion. It's basically proofreading 3'-> 5'

explain what are telomeres and why they are important for us

In eurkoytes- They protect the end of chromosomes from deterioration. No replication in that area because no genes in them. So basically a buffer zone Also prevents chromosomes from sticking to each other. Each time chromosomes replicated, telomeres gets shorter and shorter. So telomerase helps lengthen telomere s And bring them back to original length. Some cells do not have a lot of telorase and so telomeres are eliminated. So cell cannot replicate.

list and describe the 3 activities of DNA Polymerase I

In short, I think 1) goes forward (5'-3') and adds nucleotides 2) proofreads in reverse (3'-5') 3) removes and repairs forward again (5'-3') 1) A 5' → 3' (forward) DNA-Dependent DNA polymerase activity, requiring a 3' primer site and a template strand. DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of base pair synthesis by Polymerase I ~10 and 20 nucleotides/second. DNA polymerase I, is not the primary enzyme of replication; it performs DNA clean-up functions during replication, recombination, and repair. 2) A 3' → 5' (reverse) exonuclease activity that mediates proofreading. The 3′ → 5′ nuclease activity contributes to the remarkably high fidelity of DNA replication, which has an error rate of less than 10−8 per base pair (less than 1 in 108 bases is incorrect). 3) A 5' → 3' (forward) exonuclease activity involved in DNA repair and primer removal.

What phase is regulated?

Initiation phase regulated such that replication occurs only once in each cell cycle.

Topoisomerases

Introduce or eliminate supercoils by temporarily cleaving DNA Dna usually in a negative supercoil, which occurs from unwinding or underwinding of DNA. Type 1 catalyzes relaxation of supercoil (exergonic) type 2 utilize free energy of ATP hydrolysis to add neg supercoils to DNA (called DNA gyrase in bacteria)

Primers

Must have a free 3' OH bound to the template strand. The elongation starts as a nucleophilic attack by the OH on the 3rd carbon a phophodiester bridge is formed and a pyrophosphate released (you get Pi)

The synthesis of the leading and lagging strands

Polymerases synthesize the leading Andrea lagging strands simultaneously at the replication fork. DNA polymerase starts the this is of the leading strand starting from the RNA primer formed By Primase. Helicase unwind the DNA. Single-stranded binding proteins bind to the underground strand keeping the strand separated. See page 637 because the 2 core polymerases (on each strand of fork) must move together, and advance in the same direction on the DNA, the lagging strand is looped. This mode of replication has been named the trombone model because the size of the loop lengthens and shortens like the slide on a trombone

DNA polymerase 3

Principal replication enzyme More complex than poly 1 1)Very High Fidelity: a mistake is made less than once in 108 nucleotides added 2) Very high Catalytic Potency and Processivity •Catalytic Potency : 1000 nucleotides are added per second compared with only 10 per second for DNA polymerase I. This is due to the enzyme's high processivity; Processivity refers to the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate (in this case, DNA). The holoenzyme catalyzes the formation of many thousands of phosphodiester linkages before releasing its template, compared with only 20 for DNA polymerase I. The DNA polymerase III holoenzyme grasps its template and does not let go until the template has been completely replicated.

Define processivity

Processivity refers to the ability of an enzyme to catalyze many consecutive reactions without releasing its substrate (in this case, DNA). The holoenzyme catalyzes the formation of many thousands of phosphodiester linkages before releasing its template, compared with only 20 for DNA polymerase I. The DNA polymerase III holoenzyme grasps its template and does not let go until the template has been completely replicate

describe the stages of protein replication in E. coli, the proteins involved and their roles,

The entire complex of enzymes used for replication has been termed the DNA replicase system or replisome: 1) helicase seperates strands using energy from ATP hydrolysis. 2) topoisomerases - helps uncoil supercoil. And helps relieve topological stress from strand separation 3) DNA-binding proteins stabilize separated binding strands 4) primases synthesize short segments of RNA (primers) so DNA polymerase can do it's things 5) DNA polymerase I removes RNA primer And replaced by DNA 6) DNA ligases seals then nicks created when the RNA primer is removed and the gap is filled in with DNA, a nick remains in the DNA backbone in the form of a broken phosphodiester bond

What are DNA polymerase?

is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template. Key enzymes that promote the formation of the bonds joining units of the DNA backbone. They Catalyzes Phosphodiester-Linkage Formation in the 5' to 3' direction. (catalyze the step-by-step addition of deoxyribonucleotide units to a DNA strand).

Define induced fit

the change in the structure of the enzyme when it binds the correct nucleotide: This is why the binding of dNTP (deoxyribonucleoside triphosphate) to DNA polymerase induces conformational change. Makes a tight pocket for base pair consisting of the dNTP and its partner template strand.