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Topic 8: DNA Replication

Terms in this set (62)

DNA replication takes place in a _________________ manner
semiconservative
What serves as a template for synthesis?
-each strand of the double helix b/c of complementarity between the two strands
-each new strand is complementary to a parental strand
What are the 3 models for DNA replication?
1) Conservative Replication
2) Dispersive Replication
3) Semiconservative Replication
Conservative Model
-entire double-stranded DNA molecule is template for whole new molecule of DNA, and original DNA molecule is fully conserved during replication
Dispersive Replication
-both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules
-each resulting DNA molecule contains interspersed fragments of old and new DNA; none of the original molecule is conserved
Semiconservative Replication
-intermediate between conservative and dispersive
-the two nucleotide strands unwind, and each serves as a template for a new DNA molecule.
-depends on whether template is linear (eukaryotes) or circular (bacteria)
Replicon
unit of replication containing an origin of replication (where replication starts)
Bacterial DNA has a ______ origin if replication.
sinlge
Eukaryotic DNA has ______ origins of replication
many/lots
Modes of replication
1) Theta replication (in circular DNA)
2) Rolling circle replication (in circular DNA)
3) Linear eukaryotic replication (occurs at several origins of rep.)
Rolling circle Replication
-initiated by a break in one of the nucleotide strands, which exposes a 3′-OH group and a 5′-phosphate group
-new nucleotides added 3' to 5' end of broken strand
-3' end grows around in a circle
-repl. fork can produce several linked copies
-linear strand eventually cleaved from circle resulting in double-stranded circular DNA
Theta replication
-double-stranded DNA unwinds at origin of replication -> exposing single nucleotide strands that serve as templates where new DNA can be synthesized
-unwinding of double helix generates a loop, termed a replication bubble
-Unwinding may occur at one or both ends of the bubble, making it progressively larger
-DNA replication on both of the template strands is simultaneous with unwinding-bidirectional
-The point where two strands separate from the double-stranded DNA helix, is called a replication fork
-if replication bubble has replication fork at each end; process is called bidirectional replication
Bidirectional replication
two replication forks, one at each end of the replication bubble, the forks proceed outward in both directions, simultaneously unwinding and replicating DNA until they meet; produces two complete circular DNA molecules with one old and one new strand
Unidirectional replication
single replication fork is present, proceeds around entire circle; produces two complete circular DNA molecules with one old and one new strand
Linear eukaryotic replication
-occurs at several origins of replication
-At each origin DNA unwinds producing a replication bubble
-Replication takes place on both strands at each end of the bubble; two replication forks spreading outward. -eventually replication forks of adjacent replicons run into each other, and the replicons fuse to form long stretches of newly synthesized DNA
-Replication and fusion of all the replicons leads to two identical DNA molecules
Rolling-circle Replication
DNA Template: Circular
Nucleotide Strand Breakage: Yes
# of Replicons: 1
Uni or bidirectional: Unidirectional
Products: one circular molecule and one linear molecule that may circularize
Summarized Theta Replication Model
DNA Template: Circular
Nucleotide Strand Breakage: No
# of Replicons: 1
Uni or bidirectional: both
Products: two circular molecules
Linear Eukaryotic replication
DNA Template: Linear
Nucleotide Strand Breakage: No
# of Replicons: Many
Uni or bidirectional: bidirectional
Products: two linear molecules
Requirements of replication
1. A template of single-stranded DNA
2. Substrates (deoxyribonucleoside triphosphates: dNTPs)
3. Enzymes (eg: DNA polymerases) and other proteins
What is New DNA synthesized from?
deoxyribonucleoside triphosphates (dNTPs); consists of deoxyribose sugar and a base (nucleoside) attached to 3 phosphate groups
Each DNA strand grows in the ___' to ___' direction
5, 3
New nucleotides are added by DNA ______________ to the 3" OH end of growing strand
polymerase
The two strands of DNA are _______________ to eachother
antiparallel
Antiparallel
at the replication fork, one template strand is exposed in the 5' -> 3' and the other template is exposed in the 3' -> 5' direction
Since DNA replication can only occur 5'-> 3', how does it occur simultaneously on both strands?
Since the two strands are anti-parallel; DNA synthesis proceeds from right to left on unwinding strand and left to right on opposite strand
Direction of replication: Linear Eukaryotic replication
-has leading and lagging strand
-the leading strand is synthesized continuously at same direction of unwinding
-lagging strand synthesized discontinuously in direction opposite of unwinding
Bacterial Replication; Initiation in E.coli
-has circular chromosome, single origin of replication (oriC)
-initiator protein (DnaA) binds to oriC, causing unwinding of a short section of DNA (breaks H-bonds between the bases of the two strands of DNA)
Bacterial Replication; Unwinding in E.coli
-SSBs bind to exposed single strands, preventing them from reannealing until replication take place
-gyrase is a topoisomerase (controls supercoiling of DNA) makes a double stranded break ahead of replication fork to relieve tension in DNA
what does SSBs stand for?
Single strand binding protiens
Bacterial Replication; Elongation in E.coli
-to initiate DNA synthesis, primase (enzyme) synthesizes primers which are short stretches of RNA nucleotides (10-12 nucleotides long) with a 3'-OH group to whcih DNA polymerase can attach DNA nucleotides
-After primer binding, DNA polymerase elongates polynucleotide strand by catalyzing the addition of DNA nucleotides
-lagging strand loops so DNA pol. III moves along both strands simultaneously in 5'->3' direction
-conclusion: DNA must form a loop so that both strands can replicate simultaneously
E.coli has 5 different DNA polymerases
-DNA Polymerase I and III carry out DNA synthesis in replication
-other 3 function in DNA repair
DNA Polymerase I; Summarized
5'->3' Polymerase activity: yes
3' ->5' exonuclease activity: yes
5'-> 3' Exonuclease activity: yes
function: removes and replaces primers
DNA Polymerase II; Summarized
5'->3' Polymerase activity: yes
3' ->5' exonuclease activity: yes
5'-> 3' Exonuclease activity: No
function: DNA repair; restarts replication DNA halts synthesis
DNA polymerase III; Summarized
5'->3' Polymerase activity: yes
3' ->5' exonuclease activity: yes
5'-> 3' Exonuclease activity: No
function: Elongates DNA
DNA Polymerase IV; Summarized
5'->3' Polymerase activity: yes
3' ->5' exonuclease activity: No
5'-> 3' Exonuclease activity: No
function: DNA repair
DNA polymerase V; Summarized
5'->3' Polymerase activity: yes
3' ->5' exonuclease activity: No
5'-> 3' Exonuclease activity: No
function: DNA repair; translesion DNA synthesis
DNA Polymerase III
-multi-complex protein that synthesizes nucleotide strands by adding new neculeotides to 3' end of growing strand (both lagging and leading)
-5'->3' polymerase activity: allows addition of nucleotides 5'->3' direction
-3'->5' exonuclease activity: removes nucleotides in 3'->5' direction to correct errors in insertion in growing DNA (accuracy of DNA replication)
-has high processivity; can add several nucleotides to growing strand without releasing template until completely replicated
DNA Polymerase I
-like DNA pol. III, DNA pol. I has 5'->3' polymease and 3'->5' exonuclease activities (correct errors in nucleotide insertion)
-also has 5'->3' exonuclease activity; removes primers synthesized by primase and replace with nucleotides by DNA synthesis 5'->3'
-has lower processivity than DNA pol. III
-
Similarities of all of E. coli's DNA polymerases
1. synthesize any sequence specified by the template strand.
2. synthesize in the 5′→3′ direction by adding nucleotides to a 3′-OH group.
3. use dNTPs to synthesize new DNA.
4. require a 3′-OH group to initiate synthesis.
5. catalyze the formation of a phosphodiester bond by joining the 5′-phosphate group of the incoming nucleotide to the 3′-OH group of the preceding nucleotide on the growing strand, cleaving off two phosphates in the process.
6. produce newly synthesized strands that are complementary and antiparallel to the template strands.
7. are associated with a number of other proteins
DNA pol. I and DNA ligase
-RNA primers in the newly synthesized strands need to be removed and replaced with DNA.
-uses its 5' -> 3' exonuclease activity to remove the RNA primer and its 5' -> 3' polymerase activity to replace the RNA nucleotides with DNA nucleotides.
-Once all the RNA nucleotides are replaced with DNA nucleotides, a "nick" remains in the sugar-phosphate backbone of the new DNA strand, sealed by DNA ligase by forming a phosphodiester bond
Bacterial Replication; Termination in E.coli
-for some DNA molecules, its terminated when two forks meet
-in others, specific termination sequences (Ter) block further replication
-in E. coli; termination protein Tus (termination utilization substance) binds to Ter and blocks movement of helicase (stalls repl. fork)
Function of initiator protein
binds to origin and separates strnds of DNA to initiate replication
Function of DNA Helicase
unwinds DNA at replication fork
function of single-strand-binding proteins
attach to single-stranded DNA and prevent secondary structures from forming
function of DNA gyrase
moves ahead of repl. fork, making/resealing breaks in double-helical DNA to release torque that builds up as result of unwinding
function of DNA primase
synthesizes short RNA primer to provide a 3'-OH group for the attachment of DNA nucleotides
function of DNA pol. III
elongates new nucleotide strand from 3'
function of DNA polymerase I
removes RNA primers and replaces them with DNA
function of DNA ligase
joins Okazaki fragments by sealing breaks in sugar-phosphate backbone of newly synthesized DNA
Fidelity of DNA replication (= accuracy of DNA replication)
-error rate of DNA replication is low (one mistake per billion nucleotides)
- complimentarity (during DNA repl.)
-Proofreading (during DNA repl.)
-mismatch pair (after DNA Repl)
Complementarity
DNA polymerases select nucleotides based on complementarity of bases in template strand
Proofreading
-If incorrect nucleotide inserted by DNA polymerase in growing strand, the 3'-OH group of the mispaired nucleotide not properly positioned in active site of the enzyme to accept the next nucleotide.
-therefore: -Polymerization reaction stalls, and the 3' -> 5' exonuclease activity of DNA polymerase removes mispaired nucleotide and its polymerase activity inserts correct nucleotide
Mismatch repair
-incorrectly paired nucleotides remaining after repl. create deformity in secondary structure of DNA
-deformity recognized by specific enzymes, remove incorrectly paired nucleotide and replace with correct one based on complementarity to template
-enzymes must be able to distinguish between old and new strands of DNA to know which mispaired should be removed
Eukaryotic DNA replication
-replication is initiated at multiple origins
-Eukaryotic chromosomes are linear (prokayrotic chromosomes are circular)
-nucleosomes assembly of DNA (association of DNA with histones) must immediately follow DNA replication
-replication must be coordinated: timely manner, precisely and once every cell cycle
Licensing of DNA replication
-licensing of origins of replication: approval for replication early in cell cycle (G1 of interphase); complex of "replication licensing factors" binds to origin.
-In S phase, some licensing factors associate with other proteins, forming a helicase that unwinds DNA and replication initiated at licensed origins by replication machinery
-after replication initiated, licensing factors removed from DNA and prevented from binding DNA until mitosis is completed
what functions similarly in DNA replication in eukaryotes?
Eukarytoic counterparts of bacterial helicases, SSBs and topoisomerases
Different Eukaryotic DNA polymerases
-DNA pol. alpha (α)
-DNA pol. delta (δ)
-DNA pol. epsilon (ε)
-translesion DNA pol.
DNA polymerase alpha (α)
Complexes with primase (synthesizes RNA primer) and initiates DNA synthesis from primer
DNA polymerase delta (δ)
once DNA polymerase α has laid down 30-40 nucleotides, DNA polymerase δ completes replication of lagging strand
DNA polymerase epsilon (ε)
similar in function to DNA polymerase δ but replicates leading strand
Similarity of DNA polymerases delta (δ) and epsilon (ε)
high fidelity enzymes and their active sites accommodate the four nucleotide monophosphates very snugly ... abnormal bases and distorted DNA not accommodated! - if there are such lesions in template, high fidelity DNA polymerases stall and cannot bypass lesion
Translesion DNA polymerase
-low fidelity
-if lesion in template is present, this DNA pol. take over from stalled high fidelity enzymes and bypass the distortion
-active site of this DNA pol. are more open and accommodating to abnormal bases/distortions but are more error prone- may insert wrong nucleotides
-bypass lesion and continue DNA synthesis for short stretch
-detach from replication fork and high fidelity DNA polymerases take over replication
-DNA repair enzymes often repair the errors produced by translesion DNA polymerases
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