DNA replication Flashcards
How long does DNA replication take?
DNA replication in bacteria takes approx. 20 minutes, but in eukaryotes can vary from 1-4 hours in yeast to 24 hours in cultured animal cells
What were the theorized models of DNA replication?
- Dispersive- breakage and reunion
- Semi-conservative - unwind and replicate each
- Conservative- replication without unwinding
Who found evidence of the semi-conservative fashion for DNA replication?
in 1958, Meselson(Harvard )and stahl( uni of Oregon) produced evidence that DNA replicates in a semi-conservative fashion
Only semi-conservative model for DNA replication predicts experimental results
What was the experiment Meselson and stahl used to deduce the semi-conservative model?
15N is a non-radioactive heavy isotope of nitrogen , was used to tag DNA
One cell division showed equal amounts of N- 14 and N-15 while a second division showed higher amounts of N-14 than N-15
Who studied the enzymes responsible for DNA replication?
In 1959, Arthur kornberg received the Nobel prize for his research studying the enzymes responsible for DNA replication- most importantly- the identification
What is common in DNA replication across all species?
- DNA synthesized in 5’ to 3’ direction
- All four dNTPs (Deoxynucleoside triphosphate ) A C T G
- building blocks of the DNA molecule
- A fragment of DNA to act as a template
- Magnesium ions (Mg2+)
- required for DNA polymerase activity
- A primer providing a free 3’ -OH grou, most often is RNA but some organisms require DNA primer
Where does DNA synthesis begin?
At the origin of replication (ORI)
Prokaryotic genomes are usually
Circular and contain a single ORI
In E. Coli where is the only place that the replication process is regulated ?
E. Coli is only regulated at the point of initiation, therefore once the replication fork is established- replication proceeded until completion
Eukaryotic chromosomes don’t…
Have many replication origins and no replication termini
Why are there multiple origins of replications in eukaryotic genomes?
They are required to relocate the large genomes in a timely fashion, e.gl drisophili
- human haploid genome = ~3.4 million base pairs
- average size of a chromosome= 100, 000,000bp
Rate of replication= 2000 bp/minute
Therefore if there was a single origin of replication on each human chromosome it would take 830 hours 9f replication to complete
The number of origins increases with
Organisms with larger genomes
What is OriC?
The origin of replication in E. Coli has a length of 245 bp and contains two important sequences
- 13 nucleotide sequences (three in tandem array)
- 9-nucleotide sequences
What is the significance of the DNA sequences at the OriC?
The 13-mer tandem sequences at the Ori are A-T rich. Bonding between A-T is weaker than G-C. This facilitates easier melting and strand separation of the DNA molecule at this region
At the 9-nucleotide regions, DnaA initiator proteins bind to begin the process of replication
Explain the model of initiation of replication at E. coli OriC
- Multiple copies of initiator proteins (DnaA) bind to the 9mers at the origin
- Strand separation occurs at the region of the 13-mer sequences
- Helicase inhibitor (DnaC) protein delivers helicase (DnaB) to the template
- Helicase clamps around each single strand of DNA
- Helicase proceed to unwind the DNA in opposite directions away from origin
Once ORI is opened, prokaryotic elongation requires:
- DNA primase: polymerase which is required for DNA polymerase to begin elongation of DNA daughter strands
- DNA polymerase III: multi-subunit protein for elongation of DNA daughter strands, DNA synthesis of leading and lagging strands
- DNA polymerase I: removes and replaces RNA primer
- DNA ligase: ligates Okazaki fragments together
What are the main complications of elongation phase of replication in prokaryotes?
- Template dependent DNA polymerase canno5 initiate synthesis from single strand
- Both strands DNA must be copied in the 5’ to 3’ direction
- Single stranded DNA tends to re-anneal and form secondary structures
- How is DNA polymerase loaded and maintained on the single stranded DNA template, and unloaded when it reaches double stranded DNA (e.g. the next Okazaki fragment)
Why are primers needed?
DNA polymerase requires short double stranded region to provide a 3’ hydroxyl group to add nucleotides to
Explain the need for DNA primase
- in bacteria, RNA primers are synthesized by primase, a special RNA polymerase
- unrelated to transcribing DNA
- each primer 4-15 nucleotides in length and provides a free 3’ -OH group
- Once primer is completed, DNA pol III will continue synthesis of the strand
- NB —> a primer is synthesized ONCE on the leading strand, and many times on the lagging strand
Explain how DNA is replicated in a 5’ to 3’ direction
- One strand will be continuously replicated (leading strand )
- The other will be discontinuous (lagging strand) resulting in a series of short segments which need to be lighted together
The synthesis of both strands requires DNA polymerase III
Summarize the roles of DNA polymerase III
- Phosphodiester bond formation using the template strand and primer (elongation)
- Repair of base misincorporation by DNA polymerase III
What is the role of DNA polymerase III in phosphodiester bond formation ?
A) DNA polymerase catalyzes the formation of a phosphodiester bond between the 3’ OH group of the deoxyribose on the last nucleotide and the 5’ - phosphate of the dNTP precursor
B) At each step in the lengthening in the new DNA chain, DNA polymerase finds the correct precursor dNTP that can form a complementary base pair with the nucleotide on the template strand of the DNA. Pyrophosphate is liberated as a result of this incorporating a single base
Explain the ability of DNA polymerase III
A) Occassionally DNA polymerase will misincorporate a nucleotide into the growing DNA chain
B) These misincorporations need to be repaired prior to the next to be repaired prior to the next DNA replication or a mutation will occur
C) the 3’ to 5’ proofreading exonuclease activity of DNA polymerase clips off any unpaired residues at the primer terminus. It continues this activity until a base paired 3’-OH terminus is encountered
explain DNA synthesis on the lagging strand being discontinuous
Daughter DNA strands are synthesized in the 5’ to 3’ direction, therefore the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules called Okazaki fragments
In bacteria each Okazaki fragment is 1000-2000 bp in length
