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important structural features
- double helix
- nucleotides
- four bases
- hydrogen bonding between bases
- A-T & C-G
- antiparallel alignment
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semi-conservative transcription
one strand is original mother helix and the other strand is the daughter
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bacterial DNA replication
- signle origin of replication
- synthesis of new daughter strand is bidirectional
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OriC
origin of replication in bacterial chromosomes
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three types of DNA sequences in OriC
- AT-rich region
- DnaA box (4-5)
- GTAC methylation site
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the 5 DnaA box sequences serve as a binding site for DnaA protein
- DnaA proteins bind to all 4-5 DnaA boxes
- stimulates the cooperative binding of an additional 20-40 DnaA proteins to form a large complex
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witht the aid of ____ and ____, the DNA bends around the complex
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DnaA protein
- the tension caused from the wrapping of DNA around the DnaA complex causes the AT-rich region to separate
- after DnaA and DnaC proteins recruit DNA helicase to bind to site and being strand separation withing the OriC
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replication
- helicase opens helix
- DNA gryase alleviates supercoiling
- polymerase III synthesizes leading strand
- primase synthesizes RNA primer
- polymerase III elongates primer; prodcues okazaki fragments
- polymerase I excises RNA primer; fills gaps
- DNA ligase links Okazaki fragments to form continuous strand
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DNA polymerase
covalently attaches nucleotides in daughter strand
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E. coli has 5 distinct polymerases
- pol I and pol III: normal DNA replication
- pol II, IV, and V: DNA repair and replication of damaged DNA
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DNA polymerase I
- single subunit
- fills in small regions wehre RNA primers were located
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DNA polymerase III
- 10 subunits
- responsible for most DNA replication
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holoenzyme
enxyme that has many subunits to it
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two polymerase oddities
- unable to initiate DNA synthesis by linking together two individual nucleotides (primase needed)
- works only in 5' to 3' direction (no bidirectional synthesis)
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Okazaki fragments are on the _____ strand
lagging
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completion of okazaki fragments
- RNA primers removed by DNA polymerase I (5'-3')
- polymerase I fills in this region with newly synthesized DNA
- DNA ligase covalently bonds to last nucleotide made by polymerase I and the first nucleotide in the next new DNA fragment
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DNA polymerase III is a ______ enzyme
processive
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termination of sequences
- opposite OriC are termination sequences (ter sequences)
- T1 ter and T2 ter sequences work alternatively
- DNA ligase covalently links the two daughter strands, creating two circular, double stranded molecules
- topoisomerases break DNA strands and then rejoin them after strands have become unlocked
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T1 and T2 ter sequences
- T1- allows advncement of clockwise-moving forks, but prevents the movement of counter clockwise-moving forks
- T2- permits the advancement of counter clockwise-moving forks, but prevents the advancement of clockwise-moving forks
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primosome
DNA helicase and primase
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replisome
primosome and two DNA polymerase holoenzymes
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dimeric DNA polymerase
term used to describe two DNA polymerase holoenzymes that move as a unit toward the replication fork
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why does DNA replication exhibit a high degree of fidelity?
- hydroen bonds between correct partners are more stable
- induced fit phenomenon
- enzymatic removal of mismatch at 3' end of newly made strand (exonucleases)
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induced fit phenomenon
polymerase less likely to catalyze bonds between nucleotides if there are mismatched bases
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subunit epsilon comes with subunits alpha and theta to do what?
- identify and remove mismatched nucleotides
- occurs in 3' to 5' direction
- reduces error rate to 1 in 100 million
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under what optimum conditions can E. coli reproduce?
- replication must be coordinated with cell division
- mechanisms must regulate initiation of replication
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mechanism 1 of initiation
- insufficient amount of DnaA peoteins to bind to all the DnaA boxes within the OriC
- prevents premature replication
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mechanism 2 of initiation
- involves the temporary lack of adenine methylation in the GATC sites within OriC
- enzyme DAM (DNA adenine methyltransferase) methylates all the GATC sites in the newly replicated strands of DNA
- replication does not occur until after it has become fully methylated
- methyl group on parent strand but not daughter strand (hemimethylated)
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eukaryotic DNA replication
- not as well understoood
- common enzymes with prokaryotic replication
- appears to be substantially more complex
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unique features of eukaryotic replication
- multiple origins of replication
- ARS elements (Autonomously Replicating Sequences)
- origin of recognition complex (ORC)
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multiple origins of replication
- occurs bidirectionally from many origins of replication
- during S phase
- forks eventually meet and complete process
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ARS elements
- Autonomously Replicating Sequences
- ~50 base pairs
- the high percentage of A and T bases in this sequence
- consensus sequence ATTTAT (A or G) TTTA
- ^ where replication bubble begins
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origin of replicatin complex (ORC)
- 6 subunit protein complex that acts as the initiator of eukaryotic replication when it binds to the ARS in the G1 phase
- licensing factors bind to MCM
- MCM helicase (minichromosome maintenence) must coat DNA for replication to begin
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removal of RNA primers- flap endonuclease)
- DNA polymerase delta generates flap
- flap endonuclease cuts the flap off
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synthesis of more histon proteins
- needed for DNA replication
- occurs in the S phase of cell cycle
- histones are assembled into octamer structures and associate with newly made DNA very near the replication fork
- each daughter strand contains a random mixture of new and old octamers
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telomere
complex of telomeric sequences within the DNA and the special proteins that bind to it
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telomeric region
- moderately repetitive tandem arraw with a 3' overhauling region (12-16 nucleotides)
- many G's and T's
- are a problem when it comes to replication because they have more hydrogen bonds
- incomplete ends because the polymerase will not connect two nucleotides
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how to fix incomplete ends by polymerization
- telomerase recognizes teomeric sequences at the ends of chromosomes and synthesize additional repeats of telemetric sequences
- contains both proteins and RNA
- RNA sequence functions as a template allowing the attachment of a series of 6-nucleotide sequences
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telomerase and its protein component
- ribonucleoprotein
- a single RNA molecule is called TERC (telomeric RNA component)
- its protein comoponent is TERT (telomere reverse transcriptase)
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telomerase is a ____ ______
- reverse transcriptase
- sythesizes DNA from an RNA template
- only found in a few somatic cells
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