dna replication sections 8.3 -8.5 biology 391
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DNA replication Sections 8.3 -8.5 Biology 391. DNA Synthesis. Who? - all living organisms What? - making an exact duplicate of DNA When? - during S phase of interphase Where? – nucleus (or cytoplasm) How? – via several enzymes semi-conservative Denature, Anneal Primers, Extend - PowerPoint PPT PresentationTRANSCRIPT
DNA replicationDNA replicationSections 8.3 -8.5Sections 8.3 -8.5
Biology 391Biology 391
DNA Synthesis• Who? - all living organisms• What? - making an exact duplicate of DNA• When? - during S phase of interphase• Where? – nucleus (or cytoplasm)• How? – via several enzymes
– semi-conservative– Denature, Anneal Primers, Extend
• Why? – So that we can grow (divide)
DNA Structure• Sugar-Phosphate
backbone• Nitrogenous Base
– C always pairs with G– A always pairs with T– Hydrogen bonds connect
• Antiparallel – 5’ phosphate– 3’ hydroxyl
Steps of DNA replication1. DENATURE
– Binding of enzymes to existing DNA• At replication origins• Replisome: DNA and associated bound proteins
– Double helix is unwound2. ANNEALING
– 4-15 nucleotides of RNA added to ssDNA3. EXTENSION
– Synthesis of a new matching strand for each existing strand
– Leading versus Lagging strands
Enzymes Involved• Helicase – separate initial DNA strands
– SSBP – prevent reannealing of DNA strands
• Primase – lay down initial RNA primers so DNA pol III can work
• DNA polymerase I – removes RNA primers (exonuclease) and replaces with DNA (CC#1)
• DNA polymerase III – main replication enzyme• DNA Ligase – “glue” together small DNA
fragments, seal backbone
DENATURING:starting DNA replication
• Replication Origins– A-T rich; easiest to break Why?– DNA is opened by initiator proteins and helicase– Different in Euks/Proks
Eukaryotes vs. Prokaryotes• Linear chromosomes• Multiple replication
origins• Synthesis occurs in
nucleus• Only works in one
direction• DNA pol adds ~100
bases per second
• Single circular chromosome
• One origin of replication• Synthesis occurs in
cytoplasm• Bidirectional• DNA pol adds ~1000
bases per second
ANNEALING:
• DNA Pol III needs a double stranded starting point, but replication requires the DNA to become single-stranded:– Solution: Put down short, temporary starter
sequences.– Primase lays down short RNA oligonucleotides so
that DNA pol III can bind– RNA is later removed and replaced by DNA
nucleotides (via DNA pol I)
EXTENSION: adding complimentary bases
• DNA Polymerase III is major enzyme• 50 nucleotides/second/replication fork• Each strand acts as a template for making a new
double strand of DNA– Each half (strand) of the original DNA is known as a parent
strand – The new, complementary strand of DNA that matches is
called the daughter strand
DNA Polymerase is 1-directional• LEADING STRAND• Made 5’ 3’
continuously• Follows replisome as
unwound• 1 RNA primer necessary
at start
• LAGGING STRAND• Made 5’ 3’
discontinuously• Short segments =
Okazaki fragments• Goes in opposite
direction to replisome• Uses several RNA
primers• Short segments ligated
afterwards
SEMI-CONSERVATIVE REPLICATION
• ½ old DNA coding for ½ new DNA is known as SEMI-CONSERVATIVE REPLICATION
• During replication: each old double strand will:1. Unzip into 2 single strands, which will:2. Code for a complementary strand (A-T; G-C)3. Which will attach with H+ bonds to form:4. 2 new double strands with:5. 1 new single strand and 1 old single strand each
Semi-Conservative Replication• http://www.dnaftb.org/dnaftb/20/concept/index.html
FYI: Sun directly damages DNA by bonding thymine bases side-by-side. This can create confusion during replication. Polymerase hasdifficulty reading this abnormality and might skip it, match just one A or interpret as a bigpurine.
DNA Repair• DNA pol - error rate of 1 in 10,000 bases
– However proofreading minimizes this to 1 in 10mil
• Mutation any change in DNA– Causes: Genetic error, environmental factors
• Excision Repair – recognize mismatched bp, break sugar-phosphate backbone, remove. DNA pol replaces and ligase connects
Recap…• What enzyme unzips the DNA helix?• What enzyme reads and matches nucleotides
to the parent DNA strand?• When does DNA replication happen?• What is a replisome?• If the parent strand read: ATCGGGTCGT what
would the compliment be?
http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swf
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