test 2 Flashcards
G1 phase corresponds to the interval between cell division and initiation of DNA replication, cell is metabolically active and continuously grows but does not replicate its DNA>S phase (synthesis) during which DNA replication takes place>G2 phase during which proteins are synthesized in preparation for mitosis
interphase
separation of daughter chromosomes via mitosis (nuclear division) followed by cell division (cytokinesis)
M phase
cellular DNA content can be determined by incubating cells with fluorescent dye that binds to the DNA followed by analysis of fluorescence intensity of individual cells, distinguishing and sorting cells in G1, S, G2/M phases of cell cycle
flow cytometry or fluorescent-activated cell sorter (FACS)
G1=2n, S=2n to 4n, G2/M=4n until cytokinesis
DNA content in each phase
the beginning phase of mitosis, marked by the appearance of condensed chromosomes and the development of the mitotic spindle, DNA replication has already occurred, centrosomes start to move to opposite ends of the cell
prophase
chromosomes align at the equator of the spindle with one chromatid of each pair facing each chromosome
metaphase
the phase of mitosis during which microtubules begin to shorten and sister chromatids separate and move to opposite poles of the spindle
anaphase
the final phase of mitosis, during which nuclear envelope reforms, cell starts to pinch between new nuclei in preparation for cytokinesis, contractile ring of actin forms to separate cells
telophase
separate and move to opposite sides of the nucleus and serve as two poles of mitotic spindle which begin to form during late prophase and separate sister chromosomes, every cell contains 1 (2 when dividing), consists of two centrioles-defined arrangement of microtubule proteins
centrosome
DNA replication has already occurred, a second centrosome is added
late interphase
nuclear envelope breaks down (absorption into the ER), chromosomes attach to spindle microtubules via kinetochores, centrosomes move to opposite ends of the cell, cell cycle checkpoint: monitors tension on kinetichores to ensure that sister chromatids are attached to opposite poles
prometaphase
A regulatory point that prevents entry into the next phase of the cell cycle until the events of the preceding phase have been completed; integrity of the DNA is assessed at G1, proper chromosome duplication is assessed at G2 checkpoint, attachment of each kinetochore to a spindle fiber is assessed at M
cell cycle checkpoints
function to ensure damaged DNA is not replicated and passed on to daughter cells and coordinate further cell cycle progression with the completion of DNA replication or repair; function in G1, S, and G2
cell damage checkpoint
occurs towards the end of mitosis, monitors the alignment of chromosomes in the center of the mitotic spindle (metaphase plate), failure of one or more chromosomes to align properly on the metaphase plate causes mitosis to arrest at metaphase
spindle assembly checkpoint
in frog oocytes purification of the ‘cytoplasmic factor’ yielded this protein, acts as a general regulator of the transition from G2 to M; regulation by phosphorylation and dephosphorylation of Cdk1
MPF (maturation promoting factor)
in yeast cells random mutations and selection highlighted a protein kinase as essential for passing the START checkpoint, now known as a serine/threonine kinase that is a key regulator of mitosis in eukaryotic cells
CDK1
in sea urchin embryos these proteins undergo cycles of accumulation and degradation during the cell cycle, now known as member of the family of proteins that regulate the activity of Cdk’s and control the progression through the cell cycle
cyclins
regulatory subunit required for catalytic activity of the Cdk1 protein kinase, consistent with the notion that MPF activity is controlled by the periodic accumulation and degradation of this
cyclin B
cyclin B is synthesized and forms complexes with Cdk1 during G2, Cdk1 is phosphorylated at two critical regulatory positions (threonine-161 and is required for Cdk1 kinase activity and phosphorylation of tyrosine-15 and threonine-14) Phosphorylation of tyrosine-15, catalyzed by a protein kinase called Wee1, inhibits Cdk1 activity and leads to the accumulation of inactive Cdk1/cyclin B complexes throughout G2, transition from G2 to M is brought about by activation of the Cdk1/cyclin B complex as a result of dephosphorylation of threonine-14 and tyrosine-15 by a protein phosphatase called Cdc25
G2 to M transition
mitogens: stimulate cell division, growth factors: stimulate cell growth (growth factors>Ras/Raf/MEK/ERK pathway>synthesis of D-type cyclins>Cdk4, 6/CycD, survival factors: promote cell survival
type of extracellular molecules that regulate cell division
Rb (retinoblastoma) binds to transcription factor E2F and transcription is repressed, Cyclin D-Cd4k,6 dimer phosphorylates Rb releasing E2F, Rb switches E2F from a repressor to an activator of genes which encode proteins required for cell cycle progression
G1 to S transition
mediated by the activation of Cdk2/cyclin E complexes which results in part from the synthesis of cyclin E, which is stimulated by E2F following phosphorylation of Rb, repression of p27, activated CDK2 phosphorylates p27, degredation of p27, this positive autoregulation further activates Cdk2/cyclin E, which also phosphorylates and inactivates the APC/C ubiquitin ligase, preventing cyclin E degradation
progression through S phase
MCM (mini chromosome maintenance protein) and ORC (origin recognition complex) can only form a complex with DNA when MCM is unphosphorylated, cyclin E (and DDK) phosphorylate MCM which allows DNA polymerase association and DNA replication, topoisomerase knicks DNA backbone to relieve supercoiling (allows helicase activity of MCM to proceed down chromosome)
initiation of DNA replication
The ATR and ATM protein kinases are activated in complexes of proteins that recognize damaged DNA, ATR is activated by single-stranded or unreplicated DNA, and ATM principally by double-strand breaks, ATR and ATM then phosphorylate and activate the Chk1 and Chk2 protein kinases, respectively, Chk1 and Chk2 phosphorylate and inhibit the Cdc25 protein phosphatases, Cdc25 phosphatases are required to activate both Cdk2 and Cdk1, so their inhibition leads to arrest at the DNA damage checkpoints in G1, S, and G2
DNA damage checkpoints mechanism
Progression to anaphase is mediated by activation of the APC/C ubiquitin ligase, unattached kinetochores lead to the assembly of a protein complex (the mitotic checkpoint complex, MCC) that inhibits APC/C, once all chromosomes are aligned on the spindle, the inhibitory complex is no longer formed and APC/C is activated by Cdc20, APC/C ubiquitinates securin (an inhibitory subunit of a protease called separase), leading to activation of separase, separase degrades cohesin, breaking the link between sister chromatids and initiating anaphase, APC/C ubiquitinates cyclin B, leading to its degradation and inactivation of Cdk1, Cdk1 targets that were phosphorylated upon entry into mitosis are dephosphorylated, in turn driving exit from mitosis
spindle assembly checkpoint mechanism
DNA replication initiation (how replication forks form and how DNA synthesis begins), DNA replication elongation (how DNA synthesis proceeds on both strands of DNA), DNA replication termination (how replication forks come together and what happens at the end of the chromosome; base pairing enables each parent DNA strand to act as a template for the new strands of DNA to be synthesized, DNA polymerase catalyzes the formation of new DNA strands
stages of DNA replication
initiated in S phase by Cdk2/cyclin E and the DDK protein kinase, DDK phosphorylates MCM proteins resulting in binding of Cdc45, and Cdk2 phosphorylates recruiting factors that deliver GINS to the MCM hexamers converting the MCM hexamers into active CMG helicases, the CMG helicases initiate DNA replication by migrating away from the origin and forming two replication forks, during which ORC proteins dissociate from the origin
initiation of DNA replication pt. 2
clamp loading protein RFC (replication factor C) binds first, sliding-clamp protein PCNA (proliferating cell nuclear antigen) displaces RFC, polymerase binds PCNA
process for polymerase to load in initiation of replication
primase synthesizes single RNA primer on leading strand and several RNA primers on lagging strand, DNA polymerase alpha synthesized a few nucleotides linked to primer, DNA polymerase delta/epsilon elongate from where DNA polymerase alpha left off, RNaseH removes primers, DNA polymerase alpha fills in, DNA ligase connects fragments, small DNA fragments on lagging strand before ligation called okazaki fragments
initiation/elongation of replication
MCM complexes converge, SCF ubiquitin E3 ligase ubiquitinates MCM, ubiquitiniated MCM removed from DNA and targeted to P97 (segregase) to disassemble MCM complexes not degrade components
replication forks terminating
telomerase (a reverse transcriptase) expands end of template strand to avoid chromosomal shortening, primase makes RNA primer, DNA polymerase fills in; RNaseH, DNA polymerase alpha, and DNA ligase do their jobs, FEN1 (flap endonuclease 1) removes overhang
what to do at the end of the chromosome
a DNA change that results in a different amino acids being encoded
missense point mutation
a DNA change that encodes a premature stop codon
nonsense point mutation
insertion or deletion of nucleotide bases that leads to premature termination
frameshift mutation
single nucleotide base is changed and doesn’t affect amino acid
silent mutation
mismatch repair, base excision repair, nucleotide excision repair (good for fixing mutated bases); non-homologous end joining, homologous recombination (good for fixing double strand breaks)
types of DNA repair
the deaminated cytosine (uracil) is removed by glycosylase (but the sugar phosphate backbone stays intact), apyrimidic/apurinic (AP) endonuclease cleaves the DNA backbone at the site of the missing base, deoxyribosephosphodiestarase removes the sugar phosphate group from the cleaved backbone, DNA polymerase reinserts the missing base and backbone and DNA ligase rejoins the new backbone with the rest of the strand
base excision repair
DNA in E. coli is methylated (CH3 group is added) at GATC sequences, Mut proteins recognize mismatched DNA bases and recognize the unmethylated GATC sequences, MutH cleaves the unmethylated GATC sequnce making a break in the backbone of the newly-synthesized DNA strand, Mut proteins unduce an endonuclease to degrade the DNA between the strand break and the mismatch, DNA polymerase and DNA ligase work together to resynthesize the strand
strand-directed mismatch repair
excision nuclease scans for distortions in the shape of DNA (like kinks caused by thymine dimers), DNA helicase unwinds the DNA, excision nuclease cleaves the backbone upstream and downstream of the distortion, RNA polymerase synthesizes next strand section, DNA ligase repairs breaks in strand backbone
nucleotide excision repair
“quick and dirty” solution-DNA is just stuck back together by ligase, occurs most often in non-dividing somatic cells, when DNA strands are ligated back together sometimes nucleotides are missing, double strand break in DNA>end recognition by Ku heterodimers>additional proteins and processing of DNA ends>limited repair synthesis and ligation>repaired DNA has generally suffered a deletion of nucleotides
nonhomologous end joining
occurs after DNA replication but before cell division, relies on DNA strand annealing, DNA with double strand break repaired by homology-directed repair with an undamaged homologous chromosome, both strands of DNA are digested by nucleases producing two 3′ overhangs, one 3′ overhang is bound by RAD51 forming a displacement (D) loop that can be resolved by two mechanisms: (1) Synthesis-dependent strand annealing entails extension of the invading 3′ overhang using the homologous chromosome as a template, the 3′ overhang is displaced during its extension whereupon it can anneal with the opposite 3′ overhang of the DSB, the two resulting single-strand gaps can then be repaired by DNA synthesis and ligation (2) Double-strand break repair entails RAD51-mediated strand invasion by both 3′ overhangs of the DSB into the homologous chromosome, DNA synthesis and ligation yields two Holliday junctions that can be resolved with or without recombination between the homologous chromosomes
homologous recombination
a break in the genomic DNA backbone must be created for any insertion, deletion/excision, or rearrangement of the DNA to occur, the break in the DNA recruits repair machinery, the balance between DNA repair mechanisms homologous recombination and non-homologous endjoining, HR though to be “perfect” and more likely to occur in embryonic cells, NHEJ thought to be “error prone” and occurs readily in somatic cells
directed genomic modification
using two cut sites to flank a region that is desired to be deleted can cause excision of that genomic region, cleavage of single cut site will repeat until the cut site becomes mutated, mutations are usually insertion/deletion mutations (often referred to as Indel mutations
nuclease-driven mutation/excision
it works well, nucleic acid based targeting rather than protein based (each new target site does not require design of a new protein which may or may not work), high specificity given its 22bp long recognition site conferred by crRNA; natural system is from bacterial “immune system” against viruses, crRNA (gives specificity), tracrRNA, Cas endonuclease (many enzymes of this class with variable utilities), PAM sequence (signals the Cas enzyme where to cut)
CRISPR/Cas
higher efficiency conferred by use of single guide RNA rather than tracrRNA and crRNA as separate molecules, complementary region of sgRNA called spacer, system can be used in the same manner as any other nuclease
engineered CRISPR system
provided basic understanding of dominant/recessive genes, found that crossing plants of different traits yield reliable ratios
medelian genetics
incomplete dominance (intermediate traits in heterozygotes), loss of fitness associated with on allele or another (affects reproductive capacity), epigentics
Mendelian ratios that don’t add up
regulation of traits/gene expression not directly related to primary sequence of a gene (the genotype is not predictive of the phenotype)-allele penetrance, post transcriptional gene slicing, DNA methylation, chromatin structure (histones)
epigenetics
complex of eukaryotic DNA and proteins (primarily histones), chromatin can open to transcription which is called euchromatin, chromatin can be closed to transcription which is called heterochromatin
chromatin
modification on H3 associated with condensation state of chromatin; acetylation at K9, K14, K18, K23 (open to expression); phosphorylation at S10, methylation at K4, R17, R26=euchromatin, methylation of K9, K27=heterochromatin (could be opened or closed)
histone modification
histone AcetylTransferase associated with transcriptional activator (promoting open chromatin), histone DeACetylase associated with transcriptional repressor (promoting closed chromatin)
HAT and HDAC
non-covalent means to regulate chromatin status, remodeling factor binds activators and slide nucleosome to open DNA region to be transcribed, proteins that can help nucleosomes slide down to open up a region where things can bind and transcription can start
chromatin remodeling factor
in eukaryotes cytosines can be methylated at the 5 position of the ring, cytosine methylation tends to occur at CpG motifs (need a C and a G to be methylated), methylation carried out by DNA methyltransferases uses S-adenosylmethionine (SAM) as a methyl-donor, tends to be associated with transcriptional repression, particularly if the promoter region is methylated
DNA methylation
TET proteins catalyze the stepwise oxidation of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmeC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), 5fC and 5caC are removed and replaced by cytosine (C) via base-excision repair
reversal of methylation
