Genetic exam 2 - DNA replication

what are the products of DNA replication

two new daughter strands

each orginal strand serves as template

Semiconservative model

new DNA contains on parental and one daughter strand

correct model

conservative model

both parental strand stay together

dispersive model

parental and daughter segments are interspersed in both strands

Meselson and Stahl’s experiment

using light and heavy nitrogen, they could figure out the model following replication

hypothesis was semiconservative

after one generation the DNA is half heavy which is consistent with semi and dispersive

after two generations there equal light and half heavy DNA only consistent with semi

how many replication forks form in DNA replication of bacteria

2 forks form at the single origi and move bidirectionally

oriC

origin of replication in E coli

what are the important components of the origin of replication

DnaA boxes - sites for the binding of DnaA proteins

AT-rich regions - sites where the DNA strands separate

GATC methylation sites - help regulate DNA replication

Whats the sequence of events at oriC

1. DnaA proteins bind to DnaA boxes and to each other, causes DNA to bend and separate to the AT rich region

2. DnaB/Helicase binds at origin and further separates the strands traveling 5’ to 3’

Function of GATC methylation sites

DNA adenine methyltransferase (Dam) methylates on the A on both strands

initiation of replication only occurs efficiently on fully methylated DNA

DNA helicase

break hydrogen bonds between DNA strands to separate

generates positive supercoiling ahead of fork

DNA gyase travels ahead to relax

Single strand binding proteins

binds to the separated DNA strands to keep them apart

primase

makes short RNA primers which starts synthesis

DNA polymerase I

single polypeptide that remove RNA primer and replaces with DNA

DNA polymerase II, IV, V

DNA repair and replication of damaged DNA

DNA polymerase III

replicates

made of 10 subunits (alpha catalyzes bond formation)

beta subunit acts as a clamp protein allowing the protein to slide along DNA

What direction does DNA polymerase attach nucleotides

in the 5’ to 3’ direction (3’ to 5’ on OG)

leading strand

only 1 RNA primer at origin

continuous

toward fork

lagging strand

away from fork

many primers required

synthesized in okazaki fragments

forced to form a loop

DNA ligase

catalyzes the formation of a colvalent (ester) bond to connect the DNA backbones

connects okazaki fragments

primosome

DNA helicase and primase bound together

coordinates action between the two

replisome

primosome thats physically associated with two DNA polymerase holoenzymes

T1 and T2

termination sequences

T1 counterclockwise

T2 clockwise

How does termination occur in bacteria

the protein tus binds to ter sequences

catenanes

two intertwined circular molecules after DNA replication

separated by action of DNA gyrase

How are nucleotides connected chemically

DNA polymerase catalyzes formation of a covalent (ester) bond between the innermost phosphate group of incoming deoxyribonucleic triphosphate and 3’-OH of the sugar of the previous deoxynucleotide

Last two phosphates are release in pyrophosphate (PP_i)

what is the processive feature of DNA polymerase III

the enzyme remains attached tot he DNA strand

due to beta subunit which forms a dimer called the clamp protein that allows the subunits to slide freely along the DNA

keeps DNA pol III from falling off DNA after 10 nucs with a slow replication - 20 nuc per sec

makes rate 750 nucs per sec

What causes the high degree of fidelity in DNA replication

mistakes are very rare

due to

• stability of base pairing - complimentary pairs have higher stability

• structure of DNA polymerase active site - helix distortion by mismatches prevent incorrect nucleotide from fitting

• Proofreading function of DNA polymerase

How does DNA polymerase proofread

can identify a mismatched nucleotide and remove it

uses 3’ to 5’ exonuclease to digest the new strand until wrong nucleotide is removed

DNA synthesis resumes 5’ to 3’

How does DNA replication in eukaryotes start

have multiple origins of replication

replication bubbles start from each of them and merge into a completely replicated chromosome

origin of replication in yeast (simple eukaryotes)

called ARS Elements

50 bp with a lot of A and T in NFR

have a copy of ARS consensus sequence (ACS) - ATTTAT(A or G)TTTA

and B1 and B2 - enhance function, separation happens at B2

Origins of replication in more complex eukaryotes

• have G-rich sequences

  • G4 motifs - in NFR

    • forms G-quadraplex - four stranded helical molecule

• open conformation favored in flanking histones

• promoters and CpG islandsd found in NFR

What are the classes of eukaryotic origins of replication

constitutive - used all the time

flexible - used in a random manner; most common type

dormant - used during cell differentiation or only at a specific stage of development

How does the prereplication complex (preRC) form

• contains origin recognition complex (ORC)

  • acts as initiator, binds to origin

• has MCM helicase that then attaches

  • completes DNA replication licensing

  • binds to leading strands

• preRC converted to active replication site by phosphorylation via protein kinases

Polymerase alpha

replicates nuclear DNA

associates with primase

exchanges with DNA pol δ or ε required for elongation of both strands - polymerase switch

ε for leading, δ for lagging

translesion replicating polymerases

involved in replication of damaged DNA

can synthesize over abnormal region

how are primers removed in eukaryotes

polymerase δ runs into next okazaki fragments primer

pushes portion of primer into short flap

flap endonuclease removes primer

if too long Dna2 nuclease trims the long flap into short flap

whats within telomeres

moderately repetitive tandem repeats - 3’ overhang

has several guanine nucleotides

many thymine

what the replication problem with telomeres

no place for a primer on the 3’ end and DNA synthesizes 5’ to 3

end of linear chromosomes hard to replicate

telomerase has protein and RNA complentary to DNA sequence in the telomeric repeat

telomerase can bind to the 3’ overhang

lengthens sequences - binding, polymerization, tranlocation