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How does replication termination differ for linear vs. circular chromosomes
- linear- replication ends when the the polymerase reaches the telomeres
- circular-ter sites are utilized by TuS and Tus inhibits helicase
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function of telomeres
- protect the ends of the chromosome from deterioration
- prevents fusion with neighboring chromosomes
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how are telomeres synthesized
- telomerase expressed in embryonic, stem, and cancer cells
- not active in adult tissue
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DNA damage vs. mutation
physical abnormality in the DNA (ex. single/double strand breaks)
mutation is a change in the base sequence of the DNA
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hereditary diseases related to defects in DNA repair systems
- colon cancers have defects in NER repair enzymes
- Defects in NER can also lead to xeroderma pigmentosum(skin cells unable to repair UV-damaged DNA)
- Rad51 (eukaryotic homolog of Rec A) interacts with Brca 1 and 2
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3 General Post-replication DNA repair strategies
- Direct Repair
- Nucleotide Excision Repair (NER)
- Recombination repair
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Direct repair
repairs modified bases without their removal
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Example of Direct repair
- Pyrimidine dimers(cyclobutyl rings)formed by UV light
- Inhibits replication and transcription
- DNA photolysases bind and absorb a UV photo, transfer e- to break dimer
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NER
Nucleotine Excision Repair
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Example of NER
- mismatch in new DNA strand
- various systems excise the region by nicking and excising
- Pol III and ligase repair
- Dam methylase is used to methylate new correct strand
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Recombinational repair
allows damaged chromosome to be repaired using a sister chromatid or homologous chromosome as a template
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Main enzyme in Recombinational repair and role and properties
- RecA
- protein-mediated DNA synapsis
- has multiple DNA binding sites to hold a single strand and double helix together
- DNA-depedent ATPase
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what is recombinant DNA
DNA that has been formed artificially by combining constituents from different organisms
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Limitations of plasmid
generally accept <10kb of extra DNA
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main differences between bacterial plasmids and YAC's
- Yacs: used for cloning large DNA segments
- contains ARS (eukary oris), TEL, and CEN
Plasmids contains oris, but no CEN and TEL
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uses for cloned DNA
- helps detail information about entire genomes
- new avenues for identifying and isolating genes and proteins
- helps understand the function of newly discovered proteins
- helps discover new biochemical processes
- new approaches to understand metabolism
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how is reverse transcriptase used for cloning DNA
you can receive an mRNA and use RT to obtain DNA fragment
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RNA synthesis
- RNA polymerase synthesized from 5' to 3' on the antisense strand
- no primer required
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effects of transcription on DNA topology
- forms knot-dna is unwound at the leading edge of the transcription bubble and rewound at its rear
- topo I- relaxes negative super coils in front
- dna gyrase- relaxes positive supercoils in rear
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difference and similarity between eukaryotic and bacteria rna polymerases
- eukary: have separate polis for distinct RNA classes
- bacteria: only one RNAP
both have a very similar claw like structure
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Eukaryotic RNA polymerases and properties
- POL I- makes rRNA, no sensitivity to amantin
- POL II-mRNA, high sensitivity
- POL III- tRNA and snRNA, moderate sensitivity
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Steps of transcription
- binding of RNAP
- initiation of polymerization
- chain elongation
- chain termination
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Steps in Transcription Initiation step
- migration and promoter recognition
- formation of closed complex
- unwinding of DNA to form open complex
- initiation of RNA synthesis
- σ-subunit is released and RNA is elongated
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First nucleotide that attaches to RNAP to initiate synthesis
the pppGpN-OH, purine triphosphate
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how does deviation from -10 and -35 nt affect promoter activity
reduces promoter utilization
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the two mechanisms of transcription termination
- rho factor-dependent
- rho factor-independent
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differentes between two transcription termination
- rho dependent- uses rho termination factor (ATP-dependent helicase)
- Rho independent- termination sequence forms a palindrome due to run of weak A::U bonds
- allows RNA to fall off DNA
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