DNA Replication Summary

DNA REPLICATION
Anti-parallel 5OH 3PO4 & 3OH 5PO4

Phosphodiester bond sugar & phosphate
Hydrogen bonds
DNA REPLICATION FEATURES:
Semiconservative
Bidirectional
o
simultaneously unwound & elongated
Fixed origin
o
(+) consensus sequence TTTTATATTTT
Discontinuous replication
o
(+) Leading & Lagging strand, since DNA
polymerase only runs in 53 direction
TWO PHASES
1) Initiation
o
Denaturation of DNA
o
Formation of replication fork
o
Synthesis of RNA primer
2) Elongation
o
Polymerization of additional nucleotides
o
(+) phosphodiester bond - 3OH 5PO4, Mg
(coenzyme)
3) Termination
o
Re-annealing of parent strand to daughter strand
o
At INTERPHASE (S phase) 8 hours
ENZYMES:
1) Topoisomerase relieves torsional strain due to unwinding;
introduce nick/break in PD bonds
o
Topoisomerase 1 creates nick to prevent
supercoiling
2) Helicase (MCM protein) activated by cyclins
3) DNA primase
4) ssDNA binding proteins (SSBP)/ RPA prevents ssDNA
to reanneal allowing elongation
5) Exo/endonuclease corrects replication errors
6) Ligase joins adjacent DNA strands (Okazaki fragments) &
forms PD bonds in the nicks
7) DNA polymerase adds nucleotides
8) PCNA & RFC (replicating factor C) stabilizing & tethers
polymerase delta
o
RFC clamps the complex to DNA chain
9) CPK (Cyclin phosphorylated kinase) phosphorylates cyclin
DNA POLYMERASES:
Alpha
Location
Nuc,
3 5
exonucleas
e
Primase
+
Processivity
LOW
Fidelity
High
Replication
+
Repair
Beta
Nuc.
-
LOW
LOW
+
Delta
Nuc.
+
High
High
+
?
Alternating DNA & RNA sequence around 130-200

nucleotides
Multiple, separate primers BEFORE each Okazaki fragments
o
Gamma
Mito.
+
High
High
+
-
Epsilon
Nuc.
+
High
High
+
+
Alpha & Gamma (-) REPAIR

Other polymerases (kappa, etc) for DNA repair
LEADING STRAND
Starts at 3OH but synthesized in 5 3
DNA polymerase delta (+) elongation
One priming event only

LAGGING STRAND
Start at 5end but synthesized in 3 5
DNA polymerase alpha & delta (+) elongation
DNA Polymerase delta filling gaps with nucleotides
Okazaki fragments:
ELONGATION
starts with a primer of ribonucleotides, then switch to

addition of deoxyribonucleotides
forms coil around area of replication fork near polymerase

correct the direction of strand for elongation
5OH at end of growing DNA chain NUCLEOPHILE

PRIMER REMOVAL ON LAGGING STRAND
1) RNAse H1
o
Degrades RNA primer & leaves a single
ribonucleotide attached to end of the Okazaki
fragment
o
Okazaki fragments are usually 11 nucleotides long
RNAse H1 remove 10 nucleotides
2) FEN1
o
Removes the last nucleotide by peeling one or few
nucleotides forming a flap & cleaves the PD
bond at the ANGLE to release the flap
Initiation sites
Speed
Location
Mechanism
Primer
Processive enzyme
RNA removal
DNA
Prokaryotes
single
fast
cytoplasm
semiconservative
primase
Pol III
Pol I
circular
Eukaryotes
Multiple
Slower
Nucleus
Semiconservative
Pol alpha
Pol alpha & delta
Pol beta
Linear
Timing of replication activity of genes in region & NOT to

DNA sequence
Most highly compacted least acetylated regions of
chromosomes last to be replicated
Cells with SHORTER cell cycles MORE used origin of
replication sites
o
Each portion of genome should only be replicated
once/cell cycle
CYCLINS
1) Cyclin D (CDK4, 6) progression past restriction point at
G1/S boundary
2) Cyclin A, E (CDK2) initiation of DNA synthesis in early S
phase
3) Cyclin B (CDK1) transition from G2 M
TERMINATION
Telomerase adds new 6-nucleotide repeats to 3end of

telomeres
o
RNA present in telomerase base-pairs with the
overhanging 3end of telomerase and extends it by
acting both as a template & a reverse
transcriptase.
o
After copying a small number of repeats, the
complex moves down to the 3end of the overhand
and repeats the process
o
Telomerases are present at ends of linear
chromosomes
o
Telomerases expression is reactivated in tumor
cells allowing them to continue division indefinitely
without chromosome shortening
Telomeres
o
Sequence of repeated nucleotides (G or C)
present in the end of a linear chromosome
o
Maintained by telomerases
o
Do not need polymerases in base-pairing with
ribonucleotides
Undergo cycles of shortening of LAGGING starnds

due to inability of complete synthesis &
lengthening by addition of new 6 nucleotide
repeats to 3end of telomerase
REPAIR MECHANISM
DNA is the only macromolecule that is repaired than

degraded
Mismatch
repair
Baseexcision
repair
Description
Corrects errors made during
copying of DNA
Corrects single mismatch base
pair or short region of unpaired
DNA (2-5)
Solution
Methyl-directed
strand cutting
Exonuclease
digestion &
replacement
Universal repair mech.

Four sequential steps:
1)
Incision
2)
Excision
3)
Resynthesis
4)
Ligation
Base removal by Nglycosylase, abasic

sugar removal,
replacement
Prob: C, A & G spontaneously

form uracil, hypoxynthine or
Nucleotideexcision
repair
Doublestrand
break pair
xanthine, respectively
Used to replace regions of
damaged DNA up to 30 bases in
length
bulky lesion
Causes:
UV light cyclobutane
pyrimidine-pyrimidine dimers
Smoking benzopyrene
guanine (procarcinogen)
Ig-gene rearrangement
Prob: ionizing radiation,
chemotherapy, free radicals
Ku & DNA dependent-PKA
proteins combine to approx. 2
strands & unwind them
Aligned fragments form base
pairs & extra ends are removed
by nucleases & gaps are filled in,
then ligated
MISMATCH REPAIR
o
(+) LIGATION
o
Removal of a30
nucleotide oligomer
& replacement
Synapsis,
Unwinding,
Alignment
Ligation

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DNA REPLICATION

Anti-parallel 5OH 3PO4 & 3OH 5PO4

DNA REPLICATION FEATURES:

Alternating DNA & RNA sequence around 130-200

Alpha & Gamma (-) REPAIR

Starts at 3OH but synthesized in 5 3

DNA polymerase delta (+) elongation

One priming event only

Start at 5end but synthesized in 3 5

DNA polymerase alpha & delta (+) elongation

DNA Polymerase delta filling gaps with nucleotides

starts with a primer of ribonucleotides, then switch to

forms coil around area of replication fork near polymerase

5OH at end of growing DNA chain NUCLEOPHILE

Timing of replication activity of genes in region & NOT to

Telomerase adds new 6-nucleotide repeats to 3end of

Undergo cycles of shortening of LAGGING starnds

DNA is the only macromolecule that is repaired than

Universal repair mech.

Base removal by Nglycosylase, abasic

Prob: C, A & G spontaneously

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