BMB 251 1st Edition Lecture 17 Outline of Last Lecture I. ClickersII. DNA repairIII. DepurinationIV. DeaminationV. Base-excision repairVI. Nucleotide-excision repairVII. Homologous recombinationOutline of Current Lecture VIII. Homologous RecombinationIX. Strand invasion X. DNA renaturation XI. HeteroduplexXII. RecA/Rad51 proteinsXIII. Review from in-class for Exam Two materialCurrent Lecture- Clicker Question: Besides DNA polymerase, what enzyme is required for both nucleotide excisionrepair and base excision repair?o DNA ligase- Meiosis: process of making haploid sperm and egg for reproductive cells- Two homologous chromosomes have a crossover point, where a break in the double stranded DNA in BOTH homologous chromosomes are lined up perfectly so that DNA can be recombinated between each other leads to genetic diversityo Nick is introduced in both chromosomes so that the one strand can invade the other chromosome and “trade” their genetic info; only happens at certain spots on the chromosomes when chromosomes are super condensedo Proteins which stabilize this are called RecA/Rad1 by twisting around the double helices with the use of ATP- Homologous recombination: crucial for repairing double stranded breaks and other DNA damage, but also rearranges DNA sequences (can alter particular versions of gene as well as when gene is expressed)o Genetic exchange takes place between a pair of homologous DNA sequences o Many breaks in double-stranded DNA result from stalled or broken replication forkso Takes place between DNA duplexes with extensive regions of sequence similarity **Needs base pairing between a single strand from one DNA duplex and complementary strand from the otherThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Strand invasion: triggers DNA synthesis by DNA polymerase; can repair broken chromosomes with ease- DNA renaturation (aka hybridization): DNA double helix reforms from separated single strands helixes must be in open, unfolded conformations- Heteroduplex: regions of DNA helix formed when strands originate from two different DNA molecules- Several proteins act upon “freed” DNA strand which force it to invade a homologous DNA duplexo RecA protein (E. coli) and its homolog Rad51 (eukaryotes): binds tightly and in clusters tosingle stranded DNA, forming a nucleoprotein filamento RecA has multiple DNA binding sites allows it to hold together duplex DNA and single stranded DNA allows multiple synapsis reactions to occur RecA protein intertwines DNA single strand and DNA duplex Single strand then “searches” duplex for homologous sequences Strand invasion occurs Creates a heteroduplexReview- Protein activity is highly regulated; every enzyme can be turned on/off with regards to needs of cello Enzymes are either active or inactive; can go back and forth between conformations to change active siteso Can change noncovalently with an allosteric regulated (cofactor to make enzyme active; inhibitor to enzyme inactive)o Can also be activated/inactivated through phosphorylation by kinases and dephosphorylation by phosphatases (kinases and phosphatases can both either inactivate or activate enzymes, depending on activation site properties) Called a post-translational modification Some proteins have lots of phosphorylation sites; depending on how phosphorylated enzyme is can make it more and more activatedo Enzymes can also be activated when bound to GTP or inactivated when bound to GDP (called G proteins) GTP site is enzymatic and can cleave off on of GTP’s PO4 groups via hydrolysis so that it becomes inactive in its GDP-bound state To go back to GTP-bound state, exchange factor is needed to kick out GDP and add in GTP molecule- Topoisomerases: as DNA is unwound by helicase, DNA strand is put under extreme stress (like shoelaces that become more and more coiled/tangled when being pulled)o Topoisomerases (I): breaks a phosphodiester bond in the sugar-PO4 backbone of one strand so that strands can now rotate around the other (still covalently bonded) backbone to relieve all of the stress; strands are then ligated back together following thereactiono Topoisomerases (II): lets one strand of DNA slip through the other strand (by breaking the first strand and then repairing it afterwards) in order to relieve stress- Histones:o Beads on a string model: 147 nucleotide base pairs wrapped around histone complex (bead) with 50 linker nucleotide base connecters (string)o Beads coil up into the 30nm form to further condense the chromosome structureo Histone complex in each nucleosome contains 8 histone molecules (2H2A, 2H2B, 2H3, 2H4) and H1 is used as the linkers between
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