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Complete Intensive Cell Biology Notes Part 2 (4.0ed the final exam)

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Boston University
CAS BI 213

This blue: Both lecture and book mentioned notes This purple: important definitions This green: Only mentioned in lectures Chapter 6: From DNA Repair to End DNA Repair • Incorrect incorporation of bases during replication can lead to mutations o Chemical changes can also lead to mutations (spontaneous, or through radiation or chemical exposure) • Mutations can cause replication or transcription blocks ∴ high frequency of mutations • DNA repair mechanisms o Direct reversal of the chemical reaction responsible for DNA damage o Removal of the damaged bases and their replacement with newly synthesized DNA Direct reversal of DNA damage • Most DNA damage is repaired by excision of damaged base and its replacement • Direct reversal of DNA is mostly used with specific types of DNA damage that occur frequently o Pyrimidine dimers: Cyclobutane ring forms between pyrimidine bases. MUST BE ADJACENT & SAME STRAND. (Figure 6.19; Figure 6.18A)  Cyclobutane ring caused by saturation of double bonds between C 5 and C ∴6distort shape of DNA strand (creates a bump)  Blocks transcription or replication past site (bump) o O -methyl-guanine: result from exposure to UV light and alkylated guanine bases modified by addition of methyl/ethyl groups at the O (the sixth position in the sugar of pyrimidine where the O group is)  Causes guanine to base pair with thymine instead of cytosine  Alkylating agents: transfer methyl/ethyl groups to DNA base (chemical modifiers) • Direct reversal of dimers caused by UV light o Photoreactivation: energy from visible light is used to break cyclobutane ring  Pyrimidine bases remain in DNA and are restored to normal state  Common in prokaryotes and eukaryotes, BUT not present in placental mammals (i.e. humans) o Repair by enzyme O -methylguanine methyltransferase (Figure 6.20)  Transfers the methyl group from methylguanine to a cysteine residue in its active site  Restores guanine to original state  Common in prokaryotes and eukaryotes, including humans Excision repair • Most important in DNA repair in prokaryotes and eukaryotes o Damaged DNA is recognized, removed, and filled by DNA polymerase • Base-excision repair: single damaged bases are recognized and removed (Figure 6.21) o Can result from incorrect incorporation during replication o Or by deamination (removal of amine group) • Excision of base o DNA glycosylase: an enzyme that cleaves the phosphodiester bond of DNA  Yields free base and apyrimidinic site (a sugar with no base)  Recognizes deamination of adenine/cytosine, alkyline purines except O -methylguanine, and bases damaged by oxidation or ionizing radiation (only) o Apyrimidinic sites (AP sites) can form spontaneously through loss of purine bases (occurs frequently)  Repaired by AP endonuclease: cleaves adjacent to AP site o Damaged base is removed and filled in with DNA polymerase and ligase • Nucleotide-excision repair : Damaged bases are removed as part of an oligonucleotide (appx. 12-13 bases long) (Figure 6.22) o Helicase is required to remove oligonucleotide and gap is filled by DNA polymerase o No specific sequence of where to cleave o Different protein complexes accomplish this in prokaryotes than eukaryotes • Xeroderma pigmentosum (XP): cause extreme sensitivity to light and development of multiple skin cancers on regions of skin exposed to sunlight o Individuals with XP are unable to carry out nucleotide-excision repair • Repair genes identified through molecular cloning o Proteins in mammalian nucleotide-excision repair are closely related to yeast  Indicates highly conserved through eukaryotes • Mismatch repair: Identify and excise the mismatched base specifically from newly synthesized strand o Allows original sequence to be restored o E.coli (Figure 6.25)  Identifies newly synthesized DNA strand by lack of methylation of adenine in GATC sequence  Initiated by MutS (sees the mismatch); MutH (recognizes the hemimethylation) cleaves the unmodified strand; MutL (length between MutS & MutH which activates MutH)  Gap is filled by DNA polymerase and ligase o Eukaryotes (Figure 6.26)  Lack homolog of MutH ∴ not identified through DNA methylation  Strand specified to be repaired identified through single-strand breaks (Okazaki fragments) for lagging strand  Leading strand identified through growing 3’ end • Mutations in human homologs of MutS & MutL are responsible for hereditary nonpolyposis colorectal cancer (HNPCC) o Defects of genes that control MutS & MutL lead to high frequency of mutation ∴ high likelihood mutations may lead to cancer Translesion DNA synthesis • Pyrimidine dimers and other related sites of damage block DNA replication o Normal DNA polymerase cannot get past damage site o Cells possess different polymerases able to overcome sites of damage and continue replication • Translesion DNA synthesis: mechanism to bypass DNA damage at the replication fork o Can be correct damage after replication is complete • DNA polymerase V (Table above): Induced in response to UV irradiation and can synthesize across thymine dimer • Specialized DNA polymerases replace normal polymerase stuck at damaged DNA site and synthesize across the site, after which they are replaced by normal polymerases (Figure 6.27) o Low fidelity when copying undamaged DNA  However, able to insert correct bases opposite from some forms of DNA damage sites  Other forms of DNA damage not very reliable o Lack 3’—5’ proofreading ability Repair of double-strand breaks • Double-strand breaks occur naturally during replication o i.e. DNA polymerase encounters nicks at replication forks in template strand • However, double-strand breaks can occur due to ionizing radiation and chemical damage • Repair of double-strand breaks o Recombinational repair: rejoins the broken ends of the strands  Deletion of bases in surrounding site leads to high frequency of errors  Mutations in topoisomerase can lead to this (does not occur normally)  Used by cells as repair mechanism despite high error because it’s better than nothing o Homologous recombination: recombination between one damaged homologous chromosomes and an undamaged one (in eukaryotes) (Figure 6.29)  Ends of ds breaks are cut by endonuclease in 5’---3’ direction (yields overhanging 3’ single strand ends)  Single strands invade other parental molecule by homologous base pairing  Gaps are filled by repair synthesis and ligated  ONLY FUNCTIONS UNTIL METAPHASE  Leads to reassortment of genetic information between chromosomes  If not available during open window, recombinational repair will occur • Mutation in protein that recruits Rad51 (in gene BRCA2) can lead to breast cancer o Rad51 participates in homologous recombination repair Summary table. Corresponding malfunctions in repair systems with appropriate diseases. DNA Rearrangements Site-specific recombination • Site-specific recombination occurs between specific DNA sequences, usually only homologous over a short stretch of DNA o Instead of complementary base pairing, proteins target specific DNA sequences o Leads to purposeful rearrangement of DNA (role in development and gene regulation) • The immune system is an example of site-specific recombination o B lymphocytes (immunoglobulins) react with soluble antigens o T lymphocytes express cell surface proteins (T cell receptors) that react with antigens on surface of other cells o Both types of lymphocytes are able to recognize vast array of foreign antigens  Genes coding for lymphocyte antibody/ T cell receptor far exceed human genome  Are not encoded in germ line DNA, instead result of site-specific recombination of lymphocyte genes during development of immune system • Variable regions contain different amino acid sequences o Allow different antibodies to recognize different antigens and are responsible for antigen binding • Each B lymphocyte produces only a single type of antibody o Diversity of recognition occurs during site-specific recombination during B lymphocyte development o Both the light chains and heavy chains experience recombination separately from the other (Figure 6.32; Figure 6.33)  Recombination occurs by transcription of DNA followed by splicing to form different mRNA coding for each chain  Loss or gain of one to several nucleotides can result in mutations further diversifying the chains • T cell receptor (T lymphocytes) diversity occurs similarly to immunoglobulin (Figure 6.34) o α chain uses V and J segments (light chain of immunoglobulin) o β chain uses V,D, and J segments (heavy chain of immunoglobulin) o Mutations also occur during recombination • V(D)J recombination in lymphocytes (Figure 6.35) o RS sequences are present adjacent to coding sequences of individual lymphocyte genes o RAG1 and RAG2, proteins expressed in lymphocytes, attach to RS site and create a double-strand break o Strand break is joined by a nonhomologous end-joining process  Associated with loss of nucleotides o Lymphocytes contain an additional enzyme, terminal deoxynucleotidyl transferase, that adds random nucleotides to ends of DNA molecule Transposition via DNA intermediates • Transposition involves the movement of nonhomologous sequences in the genome • Transposable elements (transposons): elements that move by transposition • Via DNA intermediates (Figure 6.38) 1. Results in one copy at the new location with another copy at previous location (transposon is replicated while previous copy is being integrated) 2. Present in eukaryotes and bacteria 3. Not a useful mechanism, but promotes DNA rearrangements 4. Insertion sequences move from one chromosomal site to another without replication 1. Insertion sequences (IS): encode a gene for transposase flanked by short inverted repeats (transposase acts on these repeat sites) 2. Transposase introduces a staggered break in the target DNA (new site) and cleaves repeated sites on transposons 1. Very specific with IS sequences, but not as specific with target DNA(targets nonspecific sites) 2. Transposase joins ends of target DNA and the transposon 3. Gap in the target site is filled in by DNA synthesis and ligated • Most transposons move via nonreplicative mechanisms • Transposition via replicative methods creates purposeful DNA rearrangements that regulate gene expression 1. Transposition is initiated by a site specific endonuclease that cleaves a specific target DNA site 2. DNA is then inserted into specific site Transposition via RNA intermediates • Most transposons in eukaryotes are retrotransposons • Retrotransposons: movie via reverse transcription of RNA intermediates • Via RNA intermediates (in retroviruses) (Figure 6.39) 1. Reverse transcriptase makes a DNA copy of the viral RNA (cDNA) 2. Results in DNA molecule with long terminal repeats at both ends 1. Long terminal repeats (LTRs): repeated nucleotide sequences arising from duplicated viral RNA sites at which primers bind to initiate DNA synthesis. 2. Viral DNA integrates into host cell chromosome in a process similar to DNA transposons 3. Integration is catalyzed by integrase and occurs at many target sites 1. Integrase cleaves two bases from the ends of viral DNA and introduces staggered cut at target site DNA (similar to transposase) creating overhanging single strands 2. Overhanging ends are joined with viral DNA and gap is filled by DNA synthesis 3. Transcription of integrated provirus yields genomic viral RNA and mRNA to direct synthesis of viral proteins (all information include in transposon) 4. Genomic viral DNA is packaged into viral particles and released to infect new cells • Retrovirus-like elements also use this mechanism 1. Entirely similar to retroviruses except they are not packaged and do not spread to other cells 2. Able to move to new chromosomal sites within the same cell • Non-LTR retrontransposons are similar to retrovirus-like elements (Figure 6.41) 1. Differ in that they do not contain LTR sequences 2. LINES (long interspersed elements) 1. At 3’ end A-rich sequences derived from reverse transcription of poly-A tails added to mRNAs after transcription 2. Short direct repeats on either side of LINE ∴ staggered cut integration and repair synthesis 3. After cleavage of target DNA, the cleaved target DNA serves as a primer for reverse transcription of LINE (starts at the poly-A site, 3’) 4. Other strand is synthesized using the other cleaved DNA site and synthesizes via complementary base pairing • Unlike LINEs, short interspersed elements (SINEs) do not encode their own reverse transcriptase (Figure 6.42) 1. Structure similar to that of LINES 2. Do not encode functional RNA products 3. Responsible for pseudogenes that arose via RNA-mediated transposition (processed pseudogenes) 4. Processed pseudogenes that arose via this mechanism are recognized by their A-rich tracts and lack their introns (removed during mRNA processing) 5. Since they do not encode for their own reverse transcriptase or nucleases, other sites in the genomes might encode for them 1. i.e. other retrontransposons—LINEs • SINEs and LINEs are transposed to random sites in the cell genome 1. Lead to mutations, both positive and negative (i.e. short legs of dogs (+) or inherited human diseases (-) ) • Retrotransposons also stimulate DNA rearrangements 1. i.e. sequences of cellular DNA adjacent to LINEs are often transposed as well (leads to new combinations of regulatory/coding sequences and contributes to evolution of new genes) Gene amplification • Gene amplification: increases the number of copies of a gene • Result from repeated rounds of DNA replication of a particular region o Amplified DNA sequence can be free extrachromosomal molecule or tandem arrays of sequences • Responsible for programmed increases in gene expression • Does not occur frequently, but when it does it only happens highly specialized cells • Is not commonly used for gene regulation • An example of gene amplification is cancer cells o Increased copies of genes that direct proliferation (oncogenes) ∴ uncontrolled cell growth and tumor development Chapter 7: RNA Synthesis and Processing Transcription (Tranx) in Prokaryotes RNA Polymerase and Tranx · Central Dogma = DNA –transcription--> RNA –translation--> Protein · All cells inherit same genes, so regulation of expression is what makes one cell different from another · E. coli RNA polymerase: synthesizes RNA in 5’ to 3’ direction, like DNA pol.; has 6 subunits (Figure 7.1) o Bottom unit s = promoter recognizer; detachable from 5 core polymerase units; specialized to different conditions (e.g. starvation vs. nutrient availability) o Core pol. unit is RNA elongator o RNA pol. does not need a primer like DNA pol. Can synthesize RNA de novo at the beginning of a gene\ s subunit required for correct identification of transcription initiation sites \ can bind to DNA template and just begin replication (Figure 7.3) · Promoter: pol. binds to this sequence to start tranx of genes downstream o In prokaryotes, two sets of sequences at -10 and -35 (position relative to tranx initiation site at +1) are similar in many genes (Figure 7.2) § GC and TATA box § s subunit scans for this then rest of the polymerase comes; don’t know how pol. is signaled to join s but could be a conformation change that allows rest to bind to s subunit § These -10 and -35 promoter elements (called consensus sequences) are very important for efficient transcription o Sequence of events in tranx by E. coli RNA pol. (Figure 7.3) 1. Core + s subunit pol. binds nonspecifically to DNA template 2. Migrates along molecule upstream (toward 3’ end) until s subunit on bottom binds specifically to -10 and -35 promoter elements \ forms closed-promoter complex 3. Unwinds DNA to form open-promoter complex 4. Tranx begins 5. s subunit is released, leaving core pol. to continue transcribing 6. Core pol. moves along DNA, elongates new RNA until termination o Termination of tranx: 2 ways § Rho factor independent: GC-rich sequence forms stem loop that is too heavy for DNA to bind to \ RNA falls off (Figure 7.5) § Rho factor dependent: helicase-like protein binds to ssRNA and “unwinds” it from DNA template Repressors/Negative Control of Tranx · Prok. tranx mostly regulated at initiation, but also at elongation · Ex] only need to make b-galactosidase, the enzyme that breaks lactose down in galactose and glucose, in presence of lactose o Mechanism shown in Figure 7.7: negative control of lac operon § Operon = promoter + operator + structural genes § Operator (o) is next to tranx initiation site § i = away from operon; encodes repressor protein that blocks tranx when bound to o § Lactose not present, repressor binds to operon, tranx is blocked, b-galactosidase not produced § Lactose present, lactose binds to repressor, repressor can’t bind to operon, tranx not blocked, b- galactosidase produced o Cis-acting control elements: regulatory sequences (e.g. operator) that affect only expression of linked genes on same DNA Positive Control of Tranx · Ex] Glucose is preferred energy\ low glucose levels activate lac operon o Mechanism shown in Figure 7.8: positive control of lac operon § Lower glucose levels = higher cAMP levels § cAMP binds to CAP, a tranx regulatory protein § cAMP + CAP bind to target DNA, sequences upstream of tranx start site on lac operon § CAP interacts with a subunit of RNA pol. and helps it bind to the promoter to start tranx Eukaryotic RNA Polymerases and General Tranx Factors · Tranx more complex in euk.s than in prok.s because... 1.
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