Part Two of BMB 400
Enzymes needed for DNA replication: Chapter 5
Classes Sept 23, 25, Oct 02, 07
Covered entire chapter
Origins, terminators and control of replication: Chapter 6
Classes Oct 04, 07. Note that supercoiling is from Chapter 2, pages 77-80
(questions 2.12-2.14)
Cover all but “Cellular control of replication” pages 322-326, question 6.11,
6.18, 6.19
Mutation and Repair: Chapter 7
Class: Oct 09
Restrict coverage of mutagenesis to types of mutations and UV damage.
Not cover these topics on pages 338-348
Errors in Replication
Chemical modification by oxidation
Chemical modification by alkylation
Chemicals that cause deletions
Ionizing radiation
Cover all repair mechanisms
Not cover these questions: Questions 7.1-7.6, 7.12, 7.15-7.16.
[For questions 7.18 and 7.19a, refer to the answers to questions 7.15 and 7.16 to
see the damaged DNA to be repaired.]
Rest of Part Two
Recombination: Chapter 8
Classes: October 11, 16
Transposition: Chapter 9
Class October 18
Exam: October 21
October 07 class
• Finish replication enzymes: 2_2_repl_enzy2.pdf
– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf
• Topological problems in replication:
2_4_telom_topo_reg.pdf
– Telomerase
– Topoisomerases
– DNA supercoiling
Primase
• Synthesizes short oligonucleotides from
which DNA polymerases can begin
synthesis.
– Combination of ribonucleotides and
deoxyribonucleotides
• Does not itself require a primer.
• E. coli primase is DnaG, 60 kDa
• Acts within a large primosome.
Primers made by DnaG
• Primers can be as short as 6 nt, as long as
60 nt.
• Can substitute dNTPs for rNTPs in all
except 1st and 2nd positions
– Make hybrid primers with dNMPs and rNMPs
interspersed.
• Primase binds to CTG
– T serves as template for 1st nucleotide of
primer.
Primosome has many proteins
Pre-priming complex:
Protein
PriA
PriB
PriC
DnaT
DnaC
DnaB
gene
priA
priB
priC
dnaT
dnaC
dnaB
function
helicase, 3' to 5' movement, site recognition
needed to add DnaB-DnaC complex to preprimosome
forms complex with DnaB
helicase, 5' to 3' movement, is a hexamer
DNA dependent ATPase.
Primase = DnaG
Assay for assembly and migration of
the primosome
Convert single stranded (ss) fX174 to duplex,
replicative form (RF)
+
+
ss
RF
Steps in priming and synthesis
primer ass embly site
SS DNA
coated with
SSB
primosome
ATP
PriA
PPi's
ADP + Pi
primer synthesis
recognition step
DnaG
ATP
PriB
PriC
DnaT
prepriming
complex
NTPs
(primase)
DnaC
DnaB
ADP + Pi
Activities of DnaB and PriA in replisome
“Sewing machine” model
Control of DNA replication
Replicon
Origins and terminators
Solutions to the “end problem”
(telomeres)
Cellular control mechanisms
Oct 07 Class
• Finish replication enzymes: 2_2_repl_enzy2.pdf
– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf
• Topological problems in replication:
2_4_telom_topo_reg.pdf
– Telomerase
– Topoisomerases
– DNA supercoiling
Replicon = unit that controls replication
Replicator: cis-acting DNA sequence required for initiation;
defined genetically
Origin: site at which DNA replication initiates; defined
biochemically
Initiator: protein needed for initiation, acts in trans
Theta-form
replication
intermediates
visualized in
EM for
polyoma virus
B. Hirt
Labeling of completed DNA molecules
can map replication origins
Dana and Natahans, 1972, PNAS: map the
replication origin of SV40 by labeling
replicating molecules for
Physical map of the SV40 DNA fragments
increasing periods of time, isolatingproduced
complete
molecules,
digesting
by cleavage
with H.
influenza with
endonucleases
Hind restriction endonucleases, andrestriction
determining
which fragments have
the most radioactivity.
C
D
A
Physical map of the SV40 DNA fragments
produced by cleavage with H. influenza
restriction endonucleases
H
K
I
C
A
E
F
D
B
G
E
J
2-D gels: map number & position of replication origins
1st dimension separates by size
2nd
dimension
also
separates
by shape.
twice
unit
length
Simple Y
Fragment
size
doubles
during
replication.
"Bubble-arc"
"Y-arc"
Rela ted to dis tance
from ori to end of
fragme nt.
unit
length
Bubble
Double Y
Asymmetric
Positions of oriC and ter in E. coli
Forks meet and terminate in this approx. 100 kb region
terD and terA
block progress of
Fork 1
and
terC and terB
block progress of
Fork 2
are 23 bp binding
sites for T us, a
"contra-helicase."
E. coli chromosome
Replication fork 1
Replication fork 2
oriC
245 bp
Structure of oriC
13 13 13
9
9
9
9
• 245 bp long
– 4 copies of a 9 bp repeat
– 3 copies of a 13 bp repeat
– 11 GATC motifs
1
61
121
181
241
301
361
GGATCCGGAT
CGGGCCGTGG
AAAAGAAGAT
GCCCTGTGGA
GTGAATGATC
CTCAAAAACT
AGAGTTATCC
AAAACATGGT
ATTCTACTCA
CTATTTATTT
TAACAAGGAT
GGTGATCCTG
GAACAACAGT
ACAGTAGATC
GATTGCCTCG
ACTTTGTCGG
AGAGATCTGT
CCGGCTTTTA
GACCGTATAA
TGTTCTTTGG
GCACGATCTG
CATAACGCGG
CTTGAGAAAG
TCTATTGTGA
AGATCAACAA
GCTGGGATCA
ATAACTACCG
TATACTTATT
TATGAAAATG
ACCTGGGATC
TCTCTTATTA
CCTGGAAAGG
GAATGAGGGG
GTTGATCCAA
TGAGTAAATT
GATTGAAGCC
CTGGGTATTA
GGATCGCACT
ATCATTAACT
TTATACACAA
GCTTCCTGAC
AACCCACGAT
Initiation at oriC: Model
Termination
and
resolution
Regulation of replication by
methylation
m
GATC
CTAG
m
m
GATC
m
GATC
CTAG
CTAG
m
replicate
GATC
CTAG
m
methylate
(lags
behind
replication)
dam methylase
m
GATC
CTAG
m
Fully methylated
Hemimethylated
Fully methylated
Will replicate
Will not replicate
Will replicate
Oct 07 Class
• Finish replication enzymes: 2_2_repl_enzy2.pdf
– Primosome
• Summarize origins, terminators: 2_3_ori_ter.pdf
• Topological problems in replication:
2_4_telom_topo_reg.pdf
– Telomerase
– Topoisomerases
– DNA supercoiling