The Size and Packaging of Genomes

Transcription

1 DNA Replication

2 The Size and Packaging of Genomes Vary greatly in size Ø Smallest viruses- 4 or 5 genes Ø Escherichia coli- 4,288 genes Ø Human cell- 20,000 to 25,000 genes E. coli 4 million base pairs 1 mm long (over 1000 times larger that actual bacterial cell) DNA takes up around 10% of cell volume

3 How large is the human genome? in base pairs (bp) or billion bp 2001 papers in Nature & Science or kb kilo: 10 3 or Mbp Mega: 10 6 Formally it should be kbp (since DNA is double-stranded), but often abbreviated to kb Mb (Megabases) instead of Mbp kb (kilobases) instead of kbp How many genes are present in the human genome?

4 Bacterial genomes Plasmids - usually small (<10 kb), circular DNAs - independent replication Main chromosome (nucleoid structure) - often present in multiple copies - carry genes with functions that are nonessential to bacteria eg. antibiotic resistance virulence factors enzymes to synthesize toxins

5 DNA Structure The two polynucleotide chains of double- stranded DNA (dsdna) are held together by hydrogen bonds between base pairs, each strand can serve as a template for synthesis of the other strand (the secret of life) One side of the helix runs in the opposite direction of the other (Antiparallel) A polynucleotide chain has a 5 phosphate and a 3 hydroxyl

6 DNA Replication Replicate DNA to pass genetic info to progeny cells Process converts one parental molecule into two identical daughter molecules semi-conservative: each strand of parental molecule is template for new strand, and new molecules contain half parental and half new DNA complementary base paired

7 DNA Replication: Semi-conservative The Meselson-Stahl experiment: grow E. coli in labeled ammonium chloride and analyze the replicated double helices by density gradient centrifugation. After one round of division, results suggest that conservative replication is not occurring. After two rounds of division, banding pattern is compatible with semiconservative replication.

8 DNA Replication (ExtraChromosomal) Rolling Circle Replication Efficient mechanism for the synthesis of multiple copies of a circular genome. Used by bacteriophages DNA or the plasmid Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin (DSO). The initiator protein bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer f o r D N A s y n t h e s i s b y DNA polymerase III. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule. Also, linear copies can be converted to double-stranded circular molecules

9 Replication of the Bacterial Chromosome Initiation : recognition of an origin by a complex of proteins called the primosome (a single origin of replication in bacteria). Before DNA synthesis begins, the parental strands must be separated and stabilized in the single-stranded state forming the replication fork. Elongation: the elongation is undertaken by another complex of proteins named the replisome. Termination: At the end of the elongation, joining and/or termination reactions are necessary. Following termination, the duplicate chromosomes must be separated from one another.

10 DNA Replication: Initiation Origin of replication (oric): spans about 245 bp and includes 2 short repeat motifs: 9-nts (5 copies) and 13-nts (3 copies). The 9-nts repeat is the binding site for DnaA proteins binding in a cooperative manner until 30 copies of DnaA are bound to oric. DnaA opens up the DNA helix at a site located at one end of the oric sequence; the 13-sequence (AT rich). One initiation is completed, 2 replication forks out of the origin and progress in opposite directions; replication is bi-directional.

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12 Origin of DNA Replication General mechanism: 1. Local opening of the DNA double helix 2. Synthesis of RNA primers. 3. Synthesis of the leading strand starts from primer. 4. RNA primers start additional new DNA chains. Formation of the replication bubble.

13 Hemimethylation of the E.coli origin of replication creates a refractory period for DNA initiation. Methylation occurs at GATC sequences (11 are found in the origin of replication). 10 min after initiation, hemimethylated origins (resistant to initiation) become fully methylated by DNA methylase and become competent for initiation.

14 Initiation of Replication of the Bacterial Chromosome Binding of the initiator protein (dnaa) to the origin of replication Primosome = dnab (DNA helicase) + dnag (DNA primase) Binding of DNA helicase onto the inhibitor protein dnac Release of helicase from inhibitor by the action of dnaa and binding to DNA Helicase open the double helix and binds DNA primase (dnag) to form the primosome (dnab=dna helicase and dnag=dna primase) The synthesis of the RNA primer allows DNA polymerase to start the first chain of DNA Initiation of 3 supplementary DNA chains and formation of the replication fork.

15 DNA Replication: Initiation-Elongation Transition from initiation to elongation: once the prepriming complex is loaded onto the replication fork, the primase DnaG is recruited and loaded onto the DnaB helicase. This leads to the release of DnaC ( inhibitor protein) and allows the DnaB helicase to become active. DnaC hydrolysis ATP in order to release DnaB.

16 DNA Replication: Elongation Replication is catalyzed by the enzymes DNA polymerases and co-factors. DNA polymerase adds nts only at the 3 end of a polynucleotides chain. Primers are needed to initiate complementary strand synthesis. Only the leading DNA strand can be continuously replicated. The lagging DNA strand is replicated discontinuously. Direction: A. Leading strand, the synthesis goes in the same direction as the replication fork. B. Lagging strand, the synthesis goes in the opposite direction to the replication fork.

17 DNA Replication: Elongation The reaction of DNA replication is catalyzed by the enzyme DNA polymerase. DNA pol catalyzes the formation of a phosphodiester bond between 2 nts. DNA pol I: contains 1 subunit, has exonuclease activities: 3 to 5 and 5 to 3, involved in DNA repair and replication. DNA pol II: contains 1 subunit, involved in repair of damaged DNA rather than genome replication. DNA pol III: includes at least 10 subunits, has 3 to 5 exonuclease activity, major replicating enzyme.

18 SSB Proteins: Single-Strand Binding Proteins Single strand binding proteins (or SSB proteins) straighten the single stranded DNA (3 to 5 ) of the replicating fork and prevent the formation of hairpin structures (Stem-loop).

19 Clamp DNA polymerase has a tendency to synthesize short DNA molecules and then to quickly dissociate. This property is useful for the formation of multiple short Okazaki fragments. But this property is a handicap for the synthesis of longer DNA molecules. This is the case for the synthesis occurring on the 3 to 5 DNA strand. DNA polymerase is assisted by a clamp to help the binding to DNA until a double stranded region is encountered such as when Okazaki fragments are present. DNA polymerase then stops and the clamp is released.

20 Synthesis of Okazaki Fragments DNA synthesis in between RNA primers requires the binding and release of DNA polymerase from DNA.

21 DNA Ligase: Formation of a Phosphodiester Link

22 DNA Replication in Bacteria This figure integrates the information covered so far. Note: the association between DNA helicase and DNA primase to form a structure called primosome.

23 DNA Replication: Termination Replication terminates in a defined region; where the 2 replication forks meet at a position completely opposite to the oric. Terminator sequences bind to the protein Tus (terminus utilization substance). Tus allows a replication fork to pass if the fork is moving in one direction but blocks passes if the fork is moving in the opposite direction. Tus prevents DnaB helicase to pass. Disassembly of replication apparatus and DNA.

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25 The Overall Replication Process Requires the actions of 30 different enzymes Separate the strands Copy its template Produce two new daughter molecules