Chapter 11 Genetics Notes. Based on Brooker, 6th edition

Chapter 11: DNA REPLICATION process by which the genetic material is copied 561975676275Bacterial Chromosomes: Replication: goes in 2 directions (bidirectional) Initiation of Replication: The origin of replication in E. coli is termed oriC origin of Chromosomal replication Bacteria only divides when resources are present Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC Three types of DNA sequences in oriC are functionally significant AT-rich region (easier to separate bc only 2 H-bonds) 49149004838700DnaA boxes – bases recognized by the major groove Sequence of nucleotides that DnaA proteins binds/attaches to (open helix at A-T regions) usually repetitive GATC methylation sites (1) DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex DnaA protein binds to the chromosome at the A-T bonds – extend opening to replicate chromosomes 1) DnaA proteins help open up the helicase A) Attached to ‘boxes’ where DnaA proteins recognize and bind to certain binding sites (series of nucleotides) B) interact with each other (bind together) – 20 to 40 are necessary C) A-T region gets popped open D) DNA helicase comes in and unwinds DNA Summary: 1) Recognizes Origin of replication 2) unwinds the helicase 3) recruits helicase Ex) too much DnaA protein being made = constant replication of DNA creating more than one chromosome (2) DNA Methylation by DAM Methylase A) On an adenine of the sequence GATC, DNA Methylase adds a methyl group B) Methylation destabilizes interactions of BP and opens at the Ori this affects the binding of particular proteins Before replication: GATC sites are methylated on both strands This full methylation facilitates initiation of DNA replication After Replication: *prevents immediate replication* GATC sites are not methylated on the daughter strands This hemi-methylation does not efficiently initiate replication Several minutes will pass before Dam methylase will methylate the GATC sites in the daughter strands 29622752876550 (2) DNA Helicase 5’ 3’ Unwinds the double helix (3) Primase: RNA polymerase – acts to build small primers to initiate DNA synthesis DNA helicase and primase are physically bound to each other to form a complex called the primosome This complex leads the way at the replication fork (4) DNA polymerases: enzymes that catalyze the attachment of nucleotides to make new DNA Needs an exisiting 3’OH to add nucleotide (dNTP) – explains why Primase is needed to lay out RNA primers DNA Pol I – replaces RNA and replaces with new nucleotides Composed of a single subunit polypeptide Removes the RNA primers and replaces them with DNA 5’ 3’ exconuclease: has ability to remove nucleotides ahead of it Chews away the RNA strand and adds nucleotides (to the 3’ end) DNA Pol III Responsible for most of the DNA replication Composed of 10 different subunit polypeptides The alpha (?) subunit synthesizes DNA The complex of all 10 is referred to as the DNA pol III holoenzyme In the absence of the ? subunit (processive): DNA pol III falls off the DNA template after a few dozen nucleotides have been polymerized In the presence of the ? subunit DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides * Polymerase uses energy from incoming nucleotide (5’ 3’) and cleaves phosphate (btwn O-P)… with the release of energy its used to connect to the hydroxyl (O-H) SPONTANEOUS REACTION 3’ 5 EXCONUCLEASE: if a mistake occurs, must go back in the 3’ 5’ direction to correct error (5) DNA Ligase: uses ATP and links 3’OH and 5’P covalently BECAUSE: pols 3 & 1 cannot do this because there is not enough energy to covalently link lagging strands as only ONE phosphate is present Creates a phosphodiester bond connecting the DNA fragments to each other 43529253943350(6) DNA Gyrase: Topoisomerase – relieves the POSITIVE SUPERCOILING TERMINATION OF REPLICATION Opposite to oriC is a pair of termination sequences called ter sequences These are designated T1 and T2 T1 stops counterclockwise forks, T2 stops clockwise The protein tus (termination utilization substance) binds to these sequences It can then stop the movement of the replication forks DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) and created two intertwined molecules separated by topoisomerase Finally DNA ligase covalently links the appropriate DNA strands PROOFREADING MECHANISMS DNA replication exhibits a high degree of fidelity Mistakes during the process are extremely rare There are several reasons why fidelity is high 1. Instability of mismatched pairs A-G is too large and unstable & T-C is too small and unstable Complementary base pairs have much higher stability than mismatched pairs 2. Configuration of the DNA polymerase active site – has an INDUCED FIT (decreases error) DNA polymerase is unlikely to catalyze bond formation between mismatched pairs 3. Proofreading function of DNA polymerase (exonuclease proofreading) DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand with its 3’ to 5’ exonuclease activity MUTATION Use mutation to see what gets changed Eukaryotic DNA Replication Multiple Origins of Replication: (necessary to make replication go faster & can replicate both sides of the linear chromosome) ARS: High percentage of A –T regions Have 3 – 4 copies of specific sequence (like DnaA boxes) 1) Replication begins with assembly of the prereplication complex (preRC) ORC recognizes origin due to consensus sequence and grabs helicase Helicase – unwinds DNA 2) G1 S phase needs Kinase – adds a phosphate functional group Phosphorylation changes shape and pulls off helicase (gets released) Allows pols to continue replicating Replication switch: switches Pols ? to Pols ? (replication of DNA during S phase) Telomeres: Linear eukaryotic chromosomes have telomeres at both ends Telomeres have sequences of 1) Repetitive tandem arrays (MANY guanine & thymine nucleotides) -- TTAGGG 2) 3’ overhang that is 12 – 16 nucleotides long Telomere repeat sequences are needed because: 1) DNA Polymerase can only synthesize DNA in a 5’ 3’ direction 2) Can only elongate pre-existing strands – can’t initiate DNA synthesis PROBLEM at 3’ end of linear chromosome – end of strand can’t be replicated Thus, everytime replication occurs Chromosomes get shortened! Solution? Pack ends of chromosomes with the telomeres (repeated sequences of DNA that don’t get translated) Enzyme Telomerase prevents chromosome shortening – recognizes sequences at the end of chromosomes and synthesizes additional repeats of telomeric sequences Uses a short RNA sequence as a template for DNA synthesis produces a tandemly repeated sequence adding nucleotides to the 3’overhang end DNA polymerase comes in and base pairs to those added nucleotides Diseases can be caused when not enough junk DNA does not get added by Telomerase – then can shorten into important DNA -- causing diseases to form