MCB L1-2
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MCB L1-2 - Marcador
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| DNA of Bacteria and archaea (prokaryotes) | Circular DNA molecule is localized within the nucleoid. the majority of DNA occurs as the B form. |
| B form of DNA | 2 polynucleotide chains are in opposite orientation. Regular right handed helix. Diameter of 2nm and makes a complete turn every 3.4nm. There are 10.5 bps per turn. Flexibilities: # of bps can be altered per turn, DNA is coiled, certain features where it bends. |
| Supercoiling (in E.coli) | The DNA is supercoiled. This occurs when additional turns are introduced into the DNA double helix. Positive: with turns, Negative: turns are removed. |
| Torsional stress | It is accommodated in two ways: formation of superhelices and Altering the number of bps per turn. These responses expressed by the linking number (L): Total number of times that the two strands cross each other when constraint to lie in a plane |
| Types of Topoisomerases | Type I: Breaks one strand of DNA, pass the other strand through the gap and seal the break. L = +/- 1 (in E.coli= relaxes -ve supercoil). Type II: break both strands of the DNA, pass another part of the helix through the gap and change the L by +/- 2 (in E.coli= DNa gyrase creates -ve supercoils). |
| How is DNA organised in bacteria? | The single circular DNA molecule is organised into a series of supercoiled loops that radiate from the central protein core. The nucleoid includes: DNA gyrase and topoisomerase I that maintain the supercoiled state of the DNA, abundant HUs (heat unstable) for packaging. HUs form tetramers around which the DNA is bound (apprx 60 bps). Archea DO NOT have HUs |
| Linear and multipartite genomes | Some bacteria have a linear NOT circular genome eg. Streptomyces coelicolor. Others have multipartite genomes: Genomes divided into two or more DNA molecules. |
| How have the important components of the genome been acquired? | By horizontal gene transfer (HGT). 'Evolution in quantum leaps’. Components: 1.Prophages: Many genomes harbour phage-like elements. 2. Genomic islands (GIs): Often mutated so masking their transmission and integration modes, can confer fitness to occupy a niche. 3. Transposable genetic elements. |
| DNA replication | Pol III does most work. Pol I fills Okazaki fragments and removes RNA primers from the fragments. Leading and lagging strands. |
| Links between DNA replication and the cell cycle | Bacteria and Archaea have a cell cycle. Replicon - basic unit of replication. It is a DNA molecule with a functional origin of replication. Each replicon must be replicated at least once per cell division. 2 Links: 1. Initiation of replication commits the cell to a subsequent division and 2. Cell division cannot occur until the round of replication associated with a particular initiation has been completed. |
| Control of Initiation of Replication (E.coli) | OriC has been characterised. DNA digested with a restriction enzyme and then ligated into a plasmid lacking an origin of replication. Origin actin region is 245 bps long and contains: 1. 14 copes of GATC 2. 4/5 copies of a 9 bp seq in the right hand 2/3 oriC 3. three copies of AT-rich 13 bp seq in the left hand 1/3 of oriC. |
| Initiation in E.coli | 1. Binding of 20 monomers DNAA to the 4/5 x 9-bp repeats in the RH part of oriC, it forms a closed complex. 2. Open complex forms when the 3 AT-rich 13 bp repeats melt. 3. DNAB helicase is loaded onto the melted DNA with DNAC, ATP is hydrolysed and DNAC is released. 4. DNAB unwinds the Dna bidirectionally with SSBs and DNA gyrase. 5. Primase synthesises a primer on both strands of replication. |
| What controls whether a round of replication is initiated? | The Dam methylase methylates the adenine residues GATC motifs in OriC, which need to occur 14 times for initiation. After the replication, the new strands ae not methylated (semi conservative replication). New dna molecules are half-methylated. Re-methylation occurs about 1/3 of the way in cell cycle. |
| Termination | The two replication forks approach one another. They fuse in a region opposite to oriC, the terminus region: a ‘replication fork trap’. It has a series of DNA sites at which arrest (or pausing) of fork progression occurs. Terminator sites are polar, so arrest a fork from one direction but not the other. Tus: terminator protein used to halt the fork |
| Ter sites | The clockwise fork passes the anticlockwise ter sites (terH, I, E, D and A) but is halted at clockwise terC + Tus. If anticlockwise fork arrives replication terminates. If not the clockwise fork continues to terB where it may terminate. |
| Meselson-Radding model | Cleavage - one strand is cleaved by an endonuclease. Chain displacement - DNA synthesis displaces a chain. Invasion – The single stranded chain invades a homologous double-stranded DNA molecule. Catalysed by RecA. Chain removal – the displaced chain is digested. Ligation – produces a Holliday junction. Branch migration - increases heteroduplex, catalysed by RuvAB. Isomerization - the strands of the Holliday junction spontaneously cross and uncross, does not require catalysis. Resolution - The crossed strands of the Holliday junction cleaved by RuvC. |
| RecBCD contains: | SsDNA exonuclease (5’->3’ and 3’->5’) ssDNA endonuclease dsDNA exonuclease DNA-dependent ATPase DNA helicase |
| RecBCD steps: | 1. Binds to end of dsdna and unwinds via helicase activity. 2. Degrades both directions strands via exonuclease activity. 3. Each step is 23 bps long "quantum inch worm". 4. When meets the x-site: 3 to 5 exonuclease is inhibited, 5 to 3 exonuclease is stimulated. 5. produces ssdna 3 end tail wehre RecA binds. |
| Transposition - Inversion sequences | Dna seq can change orientation. Inversions can control gene expression |
| The hin region | 995 bps. long bounded by 2x 14 bp inverted repeats. Encodes for an invertase which catalyses inversion. |
| Structure of H1 and H2 genes | The H1 gene has it own promoter and operator and is physically separated from the hin region The H2 gene is in an operon with the rep gene that encodes a repressor for the H1 gene The promoter for the H2-rep operon lies within the hin region Phase 1: H1 expressed biut H2 worng orientation Phase 2: hin inverts, H2 correct orient., expressed and repressed H1 expression |
| DNA damage | Single base changes: produces mutations but have no effect on physical process of transcription or replication eg. replication errors, deaminations, chemical modifications of bases Structural distortions: may impede transcription and/or replication eg. Single strand breaks, Removal of a base, Covalent modification of bases. |
| Structural distortion of thymine by UV light | Two adjacent thymines on the same strand become covalently linked in a cyclobutane structure or a (6-4) photoproduct. |
| Repair mechanisms | 1. Direct Repair - reversal or simple removal of the damage. 2. Mismatch repair - detection and repair of mismatched bases. 3. Excision repair - recognition of the damage followed by excision of a patch of DNA and its replacement by undamaged DNA. 4. Tolerance systems - allow DNA replication to proceed through damaged regions of DNA. 5. Retrieval systems - recombinational processes to repair damaged DNA. |
| Direct repair: Photolyase | This process repairs any UV-induced intrastrand pyrimidine dimer. The enzyme deoxyribopyrimidine photolyase binds specifically to pyrimidine (thymine) dimers in the dark. Photolyase contains two chromophores that absorb light energy in the range 300-600 nm. Absorbed energy is used to split cyclobutane structures. |
| Mismatch repair: Uracil DNA glycosylase | Uracil is occasionally incorporated into DNA instead of thymine. Uracil DNA glycosylase removes uracil base from the nucleotide making an AP site. AP endonuclease makes a break in the phosphodiester backbone adjacent (5’) to the AP site. DNA polymerase I binds to the break and lays down new DNA and the gap is sealed by DNA ligase. |
| The mut system | MutS recognizes mismatches and short insertion/deletions on hemi-methylated DNA and binds to them. MutL binds and stabilizes the complex. The MutS-MutL complex activates MutH. MutH locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group. MutU (Helicase II) unwinds the DNA from the nick in the direction of the mismatch. DNA PolI degrades and replaces the unwound DNA and DNA ligase seals the single strand break. |
| Excision repair (E.coli) | 3 excision repair modes: Very short patch, short patch (20 bps) and long patch (1500-1000bps). Both short and long patch repair utilize the repair endonuclease. Encoded by the uvrA, uvrB and uvrC genes. uvrABC binds to damaged regions and makes an incision on both sides of the damage. UvrD separates strands. As in mismatch repair DNA pol I replaces the DNA and DNA ligase fills the gap. |
| Tolerance systems: inducible error-prone repair | Low-fidelity DNA polymerases (translesion synthesis polymerases(TSPs)) can synthesise DNA past damaged bases. Not efficient at replicating undamaged DNA accurately. Each TSP appears to have a different substrate specificity. |
| Retrieval systems: daughter strand gap repair | Does not actually repair damage. Permits replication to occur successfully. Relies on other repair processes such as excision repair to repair the damage afterwards. |
| The SOS response | If E. coli suffers severe DNA damage it activates the expression of a large number of diverse unlinked genes involved in DNA repair. All genes and operons under SOS control are subject to repression by the LexA protein. LexA has two domains – a dimerization and a DNA-binding domain. There is a conserved binding site known as a 'LexA box' located within the promoter of genes regulated by LexA. |
| The RecA protein is involved in inducing the SOS response | RecA responds, changes conformation which activates it (RecA*). sulA expression inhibits cell division. Once DNA damage is repaired RecA* converts back to RecA: LexA stops autocleaving and concentration increases and LexA represses SOS operons and cell division occurs |
| Control of transcription initiation | CONSTITUTIVE EXPRESSION - Some genes are always expressed. 2 general transcriptional mechanisms: INDUCTION - the switching on of genes when they are required REPRESSION - the switching off of genes when they are not required. Repressors are regulatory proteins which prevent transcription when bound to the DNA, activated by co-repressors. Activators are regulatory proteins which activate transcription when bound to the DNA, activated by inducers. |
| REGULONS | Genes associated with a particular physiological function may not be in just one operon. These operons may be controlled by a single regulatory protein – together they are called a REGULON. |
| Characteristics of lac mutations | The functional proteins of the Lac operon. All transcribed into mRNA, then translated to protein |
| Addiction cassettes | Produce a very stable mRNA of a lethal membrane protein. Produces ncRNA which binds to mRNA to prevent the lethal protein transcription. if plasmid in daughter cell, then continue repress lethal mRNA. If plasmid isn't present, then the lethal protein gets translated, cell death. |