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DNA metabolism
Replication
Early on - “Template” so molecules can line up in a specific order and
be joined to create a new macromolecule
1940s - DNA = genetic material
1950s - structure identified how it could act as a template for
replication and transmission of genetic info
One strand is the complement of the other
DNA metabolism
Replication Rules
Replication is
1. semi-conservative
2. bidirectional (Leading & Lagging strand synthesis)
3. Synthesized by polymerases
4. Highly accurate (proofreading)
DNA metabolism
Replication Rules
Replication is semi-conservative (each DNA strand serves as a
template for the synthesis of a new strand, producing 2 new DNA
molecules, each with one new strand and one old strand)
1957 - Meselson-Stahl
(a) Grow DNA for many generations in medium with heavy N (15N)
(b) Transfer DNA to medium with only light N (14N), after 1 gen
(c) Transfer DNA to medium with only light N (14N), after 2 gen
DNA metabolism
Replication Rules
Are parental strands completely unwound before replication? Yes
Does replication proceed in one direction or both?
Cairns
DNA metabolism
Replication Rules
Does replication begin at a unique point? Yes, called origin
DNA metabolism
Replication Rules
DNA synthesis proceeds 5’→3’ and is semidiscontinuous
How can both strands be synthesized simultaneously? 1 strand
synthesized in short fragments
bidirectional
DNA metabolism
Replication Rules
DNA is synthesized by DNA polymerases
DNA Polymerase requires
1. template (bp rules)
2. primer (short strand with free 3’-OH)
1955 - Kornberg purified and characterized DNA polymerase I from
E.Coli
DNA metabolism
Replication Rules
Accuracy of replication
High fidelity
E.Coli, 1 mistake/109 to 1010 nts added
E.Coli chromosome (~106), so error occurs once every 1000 to 10,000
replications
Discrimination between correct and incorrect nts relies on H-bonding
between correct pairs and geometry of AT and GC bp
DNA metabolism
Replication Rules
Accuracy of replication
Proofreading for mistakes
3’→5’ exonuclease activity
double checks each nt after
it is added
DNA metabolism
Stages of Replication
Initiation
Only phase that is
regulated so that
replication occurs only
once every cell cycle
DNA metabolism
Stages of Replication
Elongation
DNA metabolism
Stages of Replication
Elongation
RNA primers removed by DNA pol I (5’→3’ exo)
DNA metabolism
Stages of Replication
Elongation
DNA metabolism
Stages of Replication
Termination
Ter sequences bound by Tus
(terminus utilization
substance)
Ter-Tus halts fork
DNA metabolism
DNA Replication
Much more complicated in eukaryotes
Lots more proteins
Linear chromosomes (how replicate very ends?)
DNA metabolism
DNA Repair
DNA damage from:
1. spontaneous loss of exocyclic amino group (deamination)
C → U occurs once every 107 C residues in a day (100x a day)
A → G occurs 100x slower
2. Hydrolysis of bond between sugar and base (apurinic residue)
Occurs once every 105 purines in a day (10,000x a day)
Slower for pyrimidines
3. UV damage causes pyrimidine dimers
4. Reactive chemicals
Nitrous acid precursors
Alkylating agents (nitrogen mustard, DMS, SAM)
5. Oxidative Damage
H2O2, •OH, •O2-
DNA metabolism
DNA Mismatch Repair
Correction of mismatches increases fidelity by 100 to 1000-fold
Repairs mismatches up to
1000 bp from hemimethylated GATC
DNA Repair
DNA metabolism
Defects in genes encoding proteins involved in mismatch repair,
nucleotide-excision repair, and recombinational repair can cause cancer
Nucleotide-excision repair
sole repair pathway for pyrimidine dimers
genetic defect causes XP, xeroderma pigmentosa, these individuals are
extremely sensitive to sunlight and quickly develop sunlight-induced
skin cancer
Mismatch repair
Hereditary nonpolyposis colon cancer (HNPCC) linked to defects in
these genes
Recombinational repair
Recombination - linear sequence of DNA altered by cleavage and
rejoining of chromosome (involves RecA protein)
Repair of this type sometimes needed to reconstruct replication fork
Human breast cancer genes (BRCA1 and BRCA2) produce proteins
that interact with the human homolog of RecA, therefore these are
linked to recombination repair
10% of breast cancers have defects in BRCA1 or BRCA2
Women with defects in these genes have a >80% chance of developing
breast cancer
DNA metabolism
Stages of Replication
Elongation