chapter 5 Flashcards
DNA replication
A-T and G-C, each daughter same as parent
AT and GC how many bonds
AT- 2H (easier to break)
GC- 3 H bonds
ORC
is where replication takes place
DNA replication steps
- Initiator proteins binds to origin (AAA, ATP) (helices bind and unwind DNA)
- DNA strands separated at AT rich
- Initiator protein recruits replication proteins
– ATP (regulates) by Orc1 needed for Orc to bind to DNA
Initiation: bacteria E.coli
origin OriC has a 245bp sequence
STEPS:
1. bound to ATP, AAA+ domains of DnaA = make a spiral filament
- DnaA also recruits DnaB to the origin
- DnaB is a helicase, loaded onto the DNA by DnaC
A- unwinds (initiator)
B- recruit’s
C- opens up
Initiation: Eukaryotes
Origin is ~100bp long
- A1 and B1 sites that the ORC binds to AT-rich sites are B2 and B3- downstream
- ORC recruits Cdc6 and Cdt1, recruit the MCM helicase proteins
- make the prereplicative complex
Initiation: Single-stranded Binding Proteins
can form secondary structures that would make copying by polymerase difficult
SSB in bacteria
RPA in eukaryotes
Initiation: Helicases
must be in single not dd to replicate
STEPS:
1. Open up the helix and continuously unwinding it at the replication fork as it moves
- Bacterial helicase moves 5-3
- Eukaryotic helicase moves 3-5
Helicase, DnaB: Pro
binds ssDNA and is displaced
Steps for Eu: Helicases
- DNA helicase unwinds helix to expose ssDNA
- ssDNA coated with ssDNA binding proteins
- DNA synthesis needs a primer
Add nucleotides only to an existing 3’end
Primer is a short RNA strand synthesized by primase
Elongation
- loading sliding clam and loader
- loader of replicate polymerase
- fork movement (5-3)
Sliding clamp
- ring shape 35A
- keeps DNA polymerase to DNA
- Binds DNA polymerase through an 8 AA
- stable
Bacteria= B protein Eukaryotes= PCNA
what is a clamp loader
- 5-subunit ring structure
- Replication Factor C (RFC) in eukaryotes
- Some are ATPases
ATP-> change -> clamp loading -> spiral shape
clamp loader steps
- ATP binding
- 3 end of primer enters CL
- ATP hydrolysis, clamp closes + released
- clamp gets DNA pol
Type II: topoisomerase
STEPS:
- Both strands of DNA are cut and bound to tyrosine residues
- dsDNA segment passes through gap
- DNA re-ligated with some supercoiling relieved
Termination occurs when?
- two different forks meet
- a fork reaches the end of a linear chromosome
- polymerase meets previously replicated strand
Termination- Eukaryotes
- linear chromo
- complex= histones
Termination- prokaryotes
- circular chromsome
- less complex
- @ter site
- ter bound to tus= disassembly
makes type I1 and II topo
both termination- prokaryotes and Eu have
- three phases of replication
- Replication catalyzed by DNA polymerases
- Require helicases, single-strand binding proteins, primases, initiator proteins, topoisomerases, and ligases
DNA polymerases types
DNA polymerase III in bacteria
DNA polymerase signa and E in eukaryotes
DNA polymerase: structure
Thumb: holds elongated dsDNA
Fingers: positions incoming dNTP
Palm: connects all domains
DNA polymerase: active site
carboxylate groups (2 aspartates) coordinated with 2 magnesium
Catalyzes a phosphoryl transfer reaction by Nucleophilic attack by the
• 3ʹ-OH on the α-phosphate of incoming nucleotide
Mg: One activates the 3-OH
One interacts with dNTP to stabilize the negative leaving oxygen
DNA polymerase: Fidelity
- has precise fit in active site when base-paired with the template strand
- Mismatches = different shape and don’t fit in the active site as well
- No energy is required
•Incorrect dNTPs nevertheless sometimes added to DNA strand
Proofreading
- Polymerase moves on to next dNTP, but slows when an incorrect addition was made
- Exonuclease removes incorrect nucleotide
- Energy is required
- 3ʹ end of strand returned to polymerase active site
