EXAM I Ferguson Flashcards
What two bonds are important in the formation of DNA?
Glycosidic - links sugar to base
Phosphodiester - links bases
10bp/turn
symmetric
How many nts are distributed over how many chromosomes?
3.2x10^9 nts over 24 different chromosomes
What are banding patterns, karyotyping, and whole chromosome painting used for?
The identification and detection of chromosome numbers
Banding pattern - staining & light microscopy
Karyotyping - detect abnormalities, determine loss or gain of chromosomes
Whole Chrom. Painting
3 requirements to pass genetic information to daughter cells
OriR
Centromere - kinetochore attachment
Telomere
(ENTIRE genome is replicated; DNA serves as its own template)
What are the common interactions between DNA and histone proteins?
H.H.S. - hungry, hungry, salty!
Hydrophobic interactions
H-bonds
Salt linkages - Arginine & Lysine
What are the roles of arginine and lysine during the packing of DNA with histones?
They are (+) charges which neutralizes the (-) charge of the DNA backbone, allowing the interactions (hydrophobic, H-bonds, salt linkages) to occur
What does chromatin remodeling allow for? What enzyme is used? And does this process require energy?
Looser arrangment of histones around DNA to allow greater access for proteins
Helicase requires ATP - cuts linker DNA into fragments
Define epigenetics and provide some examples
The study of changes in organisms caused by gene expression modifications RATHER than alteration of the genetic code itself
i.e. DNA methylation, Maternal Imprinting, Histone modifications, chromatin structures
Evidence that histones are more than just structural (they’re also functional; 3 ways)
Highly conserved
DNA contains equal amounts of histone and nonhistone protein
Heterochromatin is used for gene silencing & passed on to daughter cells = Drosophila experiment (zone of inactivation) = position effect
Which amino acid(s) are important in histone modification? What enzymes add and remove it?
Lysine
Methylation of lysine via methyl transferase, removed by methyl demethylase
Acetylation of lysine via histone acetyl transferase (HATs), removed by histone deacetylase complexes (HDACs)
Serine = phosphorylated
What is the function of the code-reader complex for histone codes? List its components, what are some examples of the consequences of the histone codes? Function of code reader enzymes?
Binds histone codes on the N-terminus histone tail via covalent modifications which determines DNA packaging in nucelosomes
Components: protein modules (that bind specific histone modifications on nucleosomes), scaffold protein
Displays catalytic activity
Ex: gene silencing, gene expression, etc.
Code reader enzymes spread the marker over chromosome for continued modification
Function of barrier proteins during nucleosome/chromatin modification; example is HS4 region
T/F - remodeling is dependent upon ATP
Barrier sequences (i.e. proteins or enzymes) halt the spread of chromatin modification
HS4 region protect beta-globin locus from silencing, containing a cluster of histone acetylase binding sites
True - ATP-dependent chromatin remodeling protein - assists with condensation/decondensation
Explain the role of centromeric heterochromatin (H3) in chromatin modifications
Which set of histones are always passed on to daughter cells in forming the centromere?
- H3 histone = CENP-A that forms kinetochore (required for the attachment of mitotic spindle)
Centromere sequences contain non-required alpha satellite DNA, also found at non-centromeric positions
New centromeres can spontaneously form on fragmented DNA
- H3-H4 tetramer directly inherited
What type of chromatin is rich in gene expression? Where are heterochromatin located during gene expression?
Euchromatin
Heterochromatin = towards or inside the nucleus
Active chromatin form loops
What is the role of condensins? T/F ATP required?
Used during condensation (disentangelment of sister chromatids to allow separation for cell division), which aids the compaction via ATP hydrolysis to coil DNA into chromatids
How do genomic changes occur (genome evolution)
Via mistakes during DNA replication: (rare)
I (goto) DownTown, SD
Inversions, Translocations, Deletions, Substitutions, Duplication
Base pair substitutions
How are phylogenetic trees used for comparative genomics?
Able to compare genomes of contemporary organisms; they show differences between humans and apes
Able to determine sequence conservation
One reason for sequence conservation could be that eliminating mutations could interfere with important genetic functions = purifying selection
What is the purpose of sequence comparisons in genome evolution?
Helps to provide inside into purpose or function
5% human genome conserved, but 1.5% codes for proteins
Therefore, the human genome is highly conserved
Describe the importance of gene duplication in regards to the globin chain
Duplication and Divergence
Started with a single-chain globin that binds 1 oxygen molecule, evolution allowed for a 2nd globin chain by gene duplication followed by mutation, resulting in 4 globin chains binding 4 oxygens
Mutation gave rise beta and alpha genes; translocation = moved alpha to separate chrom.; further duplication & mutation = more specialized beta molecules (fetal, adult)
Why do germ cells and somatic cells have a high fidelity rate?
Germ-cells = to maintain the species
Somatic cells = to avoid uncontrolled proliferation/cancer
What are the key players in DNA replication?
DNA Polymerase
DNA Primase
Helicase
OriR (A-T rich)
ssDNA Binding Protein
Topoisomerase
What is the function of mismatch repair during DNA replication? What players are involved?
Corrects the errors that DNA proofreading (via 3’-5’ exonuclease) does not fix
MutS - sticks and binds to mismatch
MutL - looks for errors and triggers degradation of nicked strand
What are the proteins at the replication fork in DNA replication? ATP required?
- Sliding Clamp + Clamp loader - keeps DNA polymerase on DNA when moving and is removed once it encounters a double strand
- Assembled via ATP hydrolysis
- Stays on continuously in leading strand, new clamps are assembled on lagging strand at each Okazaki fragment
At what rate does helicase unwind DNA?
1000 nt pairs/sec
What is the role of helicase loading proteins (Cdc6 and Cdt1) and Cdks during DNA replication regulation?
Helicase loading proteins bind to ORC
Cdks removed helicase loading proteins, activating helicase and unwinding DNA and loading DNA polymerase
= S phase
How are histones disassembled (broken up into) during replication and how are they reassembled afterwards?
Disassembled = destablized into H3-H4 tetramer distributed randomly to daughter duplexes and 2 H2A-H2B dimers (1/2 old; 1/2 new) with the help of histone chaperones (chromatin assembly factors) directed by sliding clamp PCNA
Daughter strands contain a hybrid of parental and new histones = epigenetic inheritance
Explain the role of telomerase in replicating chromosome ends; what is the special sequence? Define T-loops
On the lagging strand, there is not enough space for primase to place ~10nts at the end, telomerase replenishes by elongating parental strand in 5’-3’ direction using an RNA template and DNA polymerase continues replicating lagging strand
Ensures that 3’ end is longer leaving a protruding SS end that loops back and tucks into repeat = T-loops protects ends
Shelterin - protective cap
What is caused by dyskeratosis congenita in telomeres?
Mutant telomerase RNA gene
Prematurely shortened telomeres; chromosome ends aren’t stablized and secured (T-loops)
What are the two types of DNA damage and what are the results if not repaired?
Depurination - Removing a purine (A/G) base via hydrolyzing glycosyl linkages leaving a free OH group = can result in deletion
Deamination - switching out bases resulting in base-pair substitutions; i.e. UV radiation makes covalent bonds b/w adjacent pyrimidines (T-T, C-T) = pyrimidine dimers
What are the 4 types of DNA repair pathways?
Boys Never Take (you on) Dates
Base excision repair
Nucleotide excision repair
Trancription-coupled repair
Double-stranded break repair (homologous vs. non)
Describe the mechanism of base excision repair; what are the key players
DNA glycosylases (unique for each base; i.e. uracil specific) scans and cleaves glycosyl bond connecting base w/ sugar
AP endonuclease and phosphodiesterase cut phosphodiester backbone removing the base
AP endonuclease directly repairs depurinations
Describe the mechanism of Nt excision repair; what makes it different from base excision repair?
The way damage is removed is different; phosphodiester bond is cleaved on both sides
Bulky lesions are removed i.e. chemically-induced, thymine dimers
Multienzyme complex scans double helix; Gap is repaired by DNA polymerase and ligase
What is the mechanism of transcription-coupled repair? What disease is associated with this?
Active RNA polymerase is linked with DNA repair = occurs simultaneously = urgent repair systems
RNA stays along, waits, and continues transcription by fixing the repair first
Cockayne’s Syndrome - defect in the coupled-repair
RNA polymerase is permanently stalled at sites of damage
Why are methylated cytosines problematic (DNA repair)
Deamination of methyl-C produces T mismatched with G which is recognized by a DNA glycosylase which is relatively ineffective
Occurs at some CpG sequences and is associated with inactive genes
Which DNA repair mechanism is most prominent in humans?
Non-homologous double strand break repair
Broken ends are joined and sealed via DNA ligation with a few nts being lost
Define the ATM protein that’s involved in DNA repair
Kinase that generates intracellular signals that alert the cell to DNA damage and upregulates expression of DNA repair genes
Mutation = AT; Ataxia telengiectasia - neurodegeneration, predisposition to cancer, etc.
List two ways in which double stranded breaks can occur
Homologous recombination
Nonhomologous end joining - Broken ends are joined and sealed via DNA ligation with a few nts being lost
List the steps in repairing ds stranded breaks via homologous recombination (HR) (can also repair broken or stalled replication forks)
- 5’ ends are degraded by 5’ exonuclease, leaving a 3’ overhang
- 3’ end invades homologous ds template and primes repair DNA synthesis = strand invasion
- Back home; The newly synthesized 3’ end of invading strand anneals w/ original damaged DNA via complementary base pairing
- gaps filled and ligated
Importance of RecA protein in single strand DNA pairing?
RecA directs the invading single strand to search for homologous sequence to invade and form a heteroduplex DNA = synapsis reaction
List the major differences between homologous recombination and non-homologous end joining ds break repair processes
Homologous Recombination:
- Template = daughter DNA duplex
- No loss or alteration of DNA
- Can repair other types of DNA damage, conserved
Non-Homologous End-Joining
- No template required
- Mutation created at repair site (due to overhang; flush ends being chewed by exonuclease)
- Translocations can occur
What two events occur that cause homolognous recombination?
Crossing over
Gene conversion
Occurs after meiosis and duplication on the diploid cell
When does meiotic recombination occur and what does it begin with? List the basic steps
Maternal and paternal homologous chromosomes are paired
Begins with a double stranded break
Strand invasion
Double Holliday junction
Resolution
Define Holliday Junction
DNA intermediate with 4 DNA strands from two different helices
Transient structures
Cleaved by Endonuclease (RuvC); 2 outcomes:
Crossing Over = rare
Gene Conversion = common
What occurs in each scenario of the Holliday Junction?
Left - cut at BOTH HJ (both crosses); strands separate with minimal exchange of sequences
Right - cut in opposite directions; each chromosome are swapped = crossover event
What occurs during branch migration? Does this mechanism require energy? What type of DNA does a recombination event create?
Strand invasion occurs and one strand creates a branch point and moves along the heteroduplex
Requires ATP
Recombination results in heteroduplex DNA
What rare event doesn’t allow for equal exchange of genetic material between maternal and paternal genes? When does this occur
Gene Conversion
Occurs during homologous recombination during DNA synthesis
Due to mismatch repair in heteroduplex DNA regions
What are the 3 types of transpositional recombination? List the type of Conservative site-specific recombination?
DNA-only transposons
Retroviral-like Retrotransposons
Nonretrovial Retrotransposons
Bacteriophage lambda
Define transposon; what enzyme does it encode?
Specialized DNA segments that move from one position in the genome to another
Can provide antibiotic resistance in bacteria
Encodes Transposase that allows insertion into a target DNA site
What can occur from meiotic errors?
Nondisjunction - homologs fail to separate; common during egg development at Meiosis I (most common abnormality)
Aneuploid - abnormal chromosome number
Euploid - normal chromosomes!!! :))
Define conservative site specific recombination; what are the three outcomes and what causes them to occur; how does this differ from transposition? Which microbe uses this?
Mediates rearrangements of other types of mobile DNA elements (other than transposons); Bacteriophage lambda; can be used to turn genes on/off = Cre-lox recombination
DNA integration, DNA excision, DNA inversion
EACH DNA contains special recognition sites for recombinase, no intermediates
Sites are same oritentation = integrated or excised
Inverted sites = DNA inversion
Define cohesin used in meiosis I, when do chromosomes replicate?
Cohesin - holds two sister chromatids together during S phase
Replication uccurs during S phase
Plays important role in segregating homologs during meiosis I, in meiosis II, sister chromatids are separted and cohesin breaks down
Define 3 terms that occur during Prophase I
Bivalent
Chiasma
Synaptonemal complex
Bivalent - 4 chromatin structure containing replicated maternal and replicated paternal chromosomes (2 chromosomes, 4 chromatids)
Chiasma - crossing over
Synaptonemal complex - where homologs are joined via small region of homology = pseudoautosomal region
What are the 5 phases of prophase? What occurs at each step
Little Zebras Pack Dancing Dicks
Leptotene - homologs begin to condense/pair
Zygotene - synaptonemal complex forms via transverse filaments
Pachytene - crossing-over
Diplotene - breaking down of synaptonemal complex
Diakinesis - max condensation, homologs separate, transition into metaphase
Where in the body is the sex chromosome determined during primordial germ cell development?
What gene is important for testis development?
Genital ridge
- PGCs proliferate via mitosis then meiosis to differentiate into mature haploid gametes
- SRY gene - cells diff into Sertoli cells, Sox9 gene. Sertoli cells secrete anti-Mullerian hormone and induce Leydig cel diff.
Where does spermatogenesis occur?
Seminiferous tubules - maturation moves them closer to the lumen
What are the requirements of fertilization?
Albumin - helps extract cholesterol from membrane, increasing ability to fuse w/ acrosome membrane
Ca2+, HCO3- - initiation of capacitation-associated changes
List the basic steps of fertilization
- Sperm binds to zona pellucida
- Acrosomal rxn
- Penetration thru ZP
- Fusion of plasma membranes
- Sperm contents enter egg cytoplasm; cortical rxn occurs & meiosis resumes (high [Ca2+])
Describe the cortical rxn
The release of enzymes via cortical granules from the egg to change ZP to block polyspermy
Inactivation of ZP3 so it doesn’t bind to sperm or induce acrosome rxn
