Prokaryote Replication (Gelinas) Flashcards
DNA replication occurs during which phase of the cell cycle?
S (synthesis)
Replicated DNA molecules contain:
- a parental strand and a newly synthesized strand
- parental strand serves as template for new strand
- SEMI-CONSERVATIVE
Replisome
- the complex molecular machine that carries out DNA replication
Origin of replication:
- where DNA replication is initiated
- partial opening of double helix
- region rich in A:T base pairs (less H-bonds)
- DnaA recognizes and induces “melting” of the AT-rich origin in an ATP-dependent manner
What protein recognizes and induces “melting” of the AT-rich origin in an ATP-dependent manner at the origin of replication?
DnaA
DNA strand separation is catalyzed by:
- DNA helicase (DnaB) within the pre-priming complex
- binds near the replication fork
- uses ATP to force the DNA strands apart
DNA helicase (DnaB):
- catalyzes DNA strand separation during replication
- ATP dependent
- in pre-preiming complex
Singlestranded DNA-binding (SSB) proteins:
- bind cooperatively to DNA single strands to:
- keep them apart
- protect them from nucleases
During DNA replication, what is the role of Topo II?
Works AHEAD of the replication fork to remove POSITIVE supercoiling induced by strand separation
During DNA replication, what is the role of Topo I?
Works BEHIND of the replication fork to remove NEGATIVE supercoiling
RNA primers are needed to:
- initiate DNA synthesis
- DNA polymerases cannot initiate synthesis on a totally single-stranded template
- run in 5’ to 3’ direction
RNA primers provide a ______ that serves as an acceptor of the first deoxyribonucleotide by DNA polymerase.
free 3ʼ-hydroxyl group
Primers are continuously synthesized at the replication fork on:
- the lagging strand
- only a few are needed on the leading strand
DNA polymerases only synthesize DNA in what direction?
- 5’ to 3’
- continuous on leading strand
- fragmented on lagging strand
- a lot of RNA primers
- okazaki fragments
Leading strand:
synthesized continuously in the 5ʼ to 3ʼ direction toward the replication fork
Lagging strand:
synthesized discontinuously in short 5ʼ to 3ʼ Okazaki fragments directed away from the fork.
How does chain elongation occur?
- DNA polymerase III catalyzes nucleophilic attack of the 3ʼ-OH terminus of the growing DNA chain on the innermost phosphate group of an incoming deoxyribonucleotide triphosphate.
- phosphodiester bond forms
Proofreading:
- removal of erroneously introduced nucleotides that are not complementary to the template
- done by 3’ → 5’ exonuclease activity of DNA Pol I and DNA Pol III
Mismatched nucleotides that escaped proofreading may be fixed using what pathway?
Mismatch Repair (MMR) pathway
Two functions of DNA polymerase I and III in prokaryotes:
- replication in 5’ to 3’ direction
- exonuclease activity in 3’ to 5’ direction
- proofreading
DNA chain elongation can be blocked by the incorporation of:
nucleoside analogs
(sugar group of the nucleotide has been modified to prevent further DNA chain elongation. )
RNA primer excision:
- DNA Pol I synthesizes a complementary strand until it hits a RNA primer
- DNA Pol I then uses its 5’ to 3’ exonuclease activity to excise the RNA primer
- DNA Pol I then continues to synthesize the complementary strand
- DNA Pol I can then proofread its work using its 3’ to 5’ exonuclease capability
What DNA polymerase in prokaryotes removes RNA primers?
DNA Pol I
has both 5’ to 3’ and 3’ to 5’ exonuclease activity
DNA ligase:
- covalently joins Okazaki fragments by catalyzing the ATP-dependent formation of a phosphodiester bond between 5ʼ-phosphate group and 3ʼ-hydroxyl group
