Structure of Nucleic Acid and Synthesis of DNA

Describe the organization of the genome for prokaryotes, virus, plasmids,

prokaryotes: no special subcellular structure; located in the nucleoid

Viruses: has DNA or RNA, no system for replication, transcription or translation

Plasmids: small circular DNA molecules; can replicate autonomously

Describe the organization of genome for Eukaryotes

• has a nuclear envelope

• DNA associated with Histones = chromatin

  • diffuse - euchromatin (active)

  • condense - heterochromatin (silent)

what are chromatids?

when DNA is replicated and two identical sisters are formed (chromatids)

describe the structure of DNA

nucleosides joined with inorganic phosphate

phosphodiester bond between 3’ carbon of one sugar to the 5’ carbon of the next sugar

describe the A form and Z form of DNA

A: wider right hand spiral

• occurs in dehydrated samples of DNA, in DNA-RNA hybrids, and in enzyme-DNA complexes

Z:

• linked to initiation of transcription and extensive methylation

  • z-binding protein may be invovled in regulation of transcription

what is the most common form of DNA and describe it

B-form

• axis of symmertry

• chains are antiparallel

• has major and minor grove

  • regulatory proteins, anticancer drugs and antibiotics bind here

how can strands of DNA be seperated?

• by akali

  • breaks down phosphodiester bonds in RNA

• Heat

  • converts dsDNA to ssDNA

    • this is called denaturation or melting

    • Tm = temp where 50% of dsDNA is seperated

      • depends on GC content, more GC = more stable

what is renaturation/hybridization? How is this used in different molecular biology techniques?

if temperature is slowly decreased, the ssDNA combines to dsDNA

Used in:

• Flourescent in situ hybridization (FISH): detect specific sequences

• Southern blot (DNA) and northern blot (RNA): detects sequences after seperation in gel

• PCR

in prokaryotes, how is DNA packaged?

DNA is supercoiled and attached to RNA-protein core forming nucleoid

in eukaryotes, how is DNA packaged/organized?

via binding of histones (H1, H2A, H2B, H3, H4)

nucleosome core formed via 8 histones molecules;

nucleosome combine to produce tubular structure called Solenoid (30 nm fiber)

these 30 nm fiber (aka chromatin fiber) is then organized into loops and anchored via nuclear scaffold proteins

linker dna = histone 1

Describe the location of these chromosomes in humans

it is found that each individual chromosomes occupy different locations in the cell nucleus,

what are the five posttranslation modification on Histones

1. acetylation

2. methylation

3. ubiquination

4. sumoylation

5. phosphorylation

what does acetylation do to nucleosomes?

acetylation of lysinee → decreased net positive charge→ decreases histone-DNA interaction → open chromatin;

these are done by HATs (histone acetyltransferase) which coactivator proteins have. These are activated by positive transcription factors

describe histone methylation

histone methyltransferases methylates H3 on lys4, 9, 27 and 79.

methylation have different biological effects

what are genetic locus and alleles?

genetic locus: position of a gene on a chromosome

alleles: alternative versions of a gene found on different homologous chromosomes

what is karotyping?

technique used to determin the diploid number of chromosomes

what is turner syndrome?

• single x chromosome in females

• universally infertile

• kidney/cardiovascular problems

• lack of estrogen = osteoporosis

What is Klinefelter syndrome?

• most common male disease: XXY

• infertile, gynecomastia (enlargment of breast tissue in males)

• germ cell/ male breast cell cancer

What is edwards disease?

• trisomy of chromosome 18

• Kidney malformations and structural heart defects.

• Intestines protruding outside the body and developmental delays

What is down syndrome?

• trisomy of chromosome 21

What is patar syndrome?

• trisommy of chromosome 13

• Can results in microcephaly, heart and kidney defects, and abnormal

  gemitalia

list two RNA molecules that can catalyze reactions

• RNAase P: cleaves off extra RNA sequences at end of tRNA

• peptidyl transferase: activity of ribosome

what is the difference between prokaryotic and eukaryotic ribosomes

• Prokaryotes: contain only three types of rRNA and less proteins.

• Cytoplasmic eukaryotic ribosomes contain four types of rRNA and more proteins.

describe snRNA

• A component of small nuclear ribonucleoproteins (snRNPs or
  “snurps”).

• Involved in maturation of RNAs precursors - splicing

describe miRNA (microRNA)

• Small non-coding RNAs ~20-24 nucleotides

• Derived from endogenous genes

• Repress protein biosynthesis, usually by preventing the ribosome

• binding to mRNA or promotes mRNA degradation.

describe long noncoding RNA (LncRNA)

• 200 bp long

• Epigenetic modifications of DNA

• Example: Xist RNA (X-inactivation specific transcript) inactivates one of the two X chromosomes in vertebrates.

describe Large intergenic non-coding RNAs (lincRNAs)

• >200 bp long

• Example: scaffold DNA-chromatin complexes

• lincRNA HOTAIR is overexpressed in breast tumors

how can DNA synthesis be inhibited and give examples

by incorporation of certain nucleotide analogs.

• Replacement of hydroxyl group by azido group Zidovudine (AZT)

  • reverse transcriptase has higher affinity for AZT then normal human DNA polymerase

• Removal of hydroxyl group from deoxyribose Didanosine (ddI)

• Conversion of deoxyribose to another sugar, arabinose.

what does Azacitidine (Vidara) do?

• chemical analogue of cytidine

• Used to treat myelodysplastic syndrome.

• Can be incorporated into RNA and DNA.

  • In RNA, it interferes with polyribosome assembly and tRNA recognition

  • In DNA, it covalently binds to DNA methyltransferases, which interferes with DNA synthesis and triggers DNA repair and degradation of methyltransferase.

• Recently showed promising results in the treatment of gliomas

What does 5’- flourauracil do? (5-FU)

inhibits the synthesis of the thymine
nucleotides

5-FU converst dUMP to F-dUMP which prevents dUMP → dTMP

What is semiconservative replication?

in DNA replication: one strand is parental, the other

is a newly synthesized.

Describe the origins of replications in prokaryotes and eukaryotes

prokaryotes: replication begins at single, unique origin of replication.

eukaryotes: replication begin at several sites (AT-rich sites)

Describe the Pre-priming complex and their subunits

Unwinds double helix ahead of advancing replication fork and keep strands separated.

consist of three enzymes:

1. DNA helicase unwinds double helix.

2. DnaA protein binds and melts dsDNA.

3. ssDNA-binding (SSB) proteins bind to ssDNA.

   • keeps strand separate and protect them from nucleases

how do we prevent supercoils?

DNA topoisomerases remove supercoils and

facilitates transcription.

Describe the direction of DNA replication. Differentiate between leading strand and lagging strand

• DNA polymerases read DNA sequence in 3’→5’ direction, and synthesize DNA strand in 5’→3’ direction.

• Leading strand is being copied in the direction of replication fork = Synthesized continuously

• Lagging strand: being copied in the direction away from the replication fork = not continuous

  • okazaki fragments: small frag. of DNA being created near replication fork

Describe the initiation and elongation step of DNA replication

1. primases are specific RNA polymerases that synthesize RNA primers

2. DNA pol III uses RNA primer to initiate replication. Elongation is done by this too

   • also major enzyme used for proofreading

For lagging strand

• DNA Pol III synthesize until it is blocked by a RNA primer; then, it moves to another primer to synthesize new Okazaki fragment.

3. DNA Pol I replaces RNA primers with DNA.

4. Ligases join the ozarki fragments together

   • also responsible for DNA repair and recombinant DNA techniques

   • Ligases joins the fragments by forming phosphodiester bond between 3’ hydroxyl end of one nucleotide and 5’ phosphate end of

     another nucleotide. (Requires ATP)

What are the functions of Pol I, Pol II and Pol III

what are the two differences in DNA replication between eukrayotes and prokaryotes

Eukaryotes

1. Multiple origins of replication

2. RNA primers are removed by RNAse H

List out the different functions of the DNA polymerases used by eukaryotes

why does DNA shorten after every replication? why is this a problem?

There is no way to fill the gap after the removal of

RNA primer from extreme 5’-end of the chromosome

Consequence: loss of genetic information and is associated with aging,

What is telomeres? Their functions? What are telomerases?

at the end of DNA, contains telemers which are region of highly repetitive DNA at the end of chromosome (TTAGGG for humans)

function:

1. Serve as a buffer, which is consumed during cell division

2. Protect the ends from nucleases

3. Limit cells to a fixed number of divisions, in most somatic cells

• Can be replenished by telomerase

  • Active in germ, stem, and cancer cells

How do we maintain our telomers?

excercise upregulated telomerase in aorta and mononuclear cells in mice,

Higher vitamin D level is associated with longer telomere lenght in leukocytes,

Describe the mechanism of telomerase

• RNA-dependent DNA polymerase

• Contains RNA which is complementary to repetitive sequence in telomere

• Uses 3’ end of DNA as a primer and RNA as a template to extend 3’ end of DNA

• Once a single-stranded portion of telomere end is extended, an RNA primer is produced to fill a single-stranded portion of chromosome.

What is TERC?

Telomerase RNA component (TERC), is a component of telomerase used to extend

telomeres. TERC serves as a template for

telomere replication (reverse transcription) by

telomerase

what are some health functions disrupted by short telomers/telomerase

shorter = more hematopoisis

telomerase = a contributor to uncontrollable, indefinite growth (HeLa Cells)

what are possible causes of DNA mutations

1. mutagens

   • Chemicals produced by cells

   • Chemicals from environment

   • Radiation: X-rays and UV light

2. Carcinogens

3. mismatches; without adequate repair mech = mutation

describe how X-rays cigarettes smoke and UV light can cause DNA damage

1. X-Rays: produces free radicals from H2O.

2. cigarette smoke contains carcinogen benzo[a]pyrene

   • When oxidized in the body, it forms adducts with guanine residues.

3. UV light forms covalent dimer
   between adjacent thymines on
   DNA strands.

describe Xeroderma pigmentosum

• Autosomal recessive

• It is caused by genetic defect in nucleotide excision repair (NER) system.

• Cells cannot repair damaged DNA leading to skin cancer.

Describe the four repair mechanism: direct reversal pair, NER, base excision repair, and double strand break repair

direct reversal pair:

• E.coli removes thymine dimer by photactivated enzymes.

• They cleave bond between bases using energy from light

nucleotide exclusion repair:

• Endonuclease cleaves abnormal chain and remove distorted region.

• Gap is filled by DNA polymerase.

• Ends are joined by DNA ligase

Base excision repair:

• DNA glycosylases recognize distortion and cleaves N-glycoside bond.

• AP endonuclease cleaves sugar-phosphate strand at this site.

• Same types of enzymes that are involved in other repair mechanisms finish the repair.

Double-strand break repair:

• Repaired through homologous recombination.

• Homologous chromosome is used as template to repair DNA.

• Mutations in BRCA1 gene, a component of homologous recombination repair, result in an increased risk of breast cancer.

what four similarities connects the various repair mechanism mentioned?

1. Recognition of distortion.

2. Removal of the region with distortion.

3. Replacement of the gap by DNA polymerase.

4. Joining the ends by DNA ligase.

how does the cell know which strand is damaged and needs to be repaired?

in bacteria: only adenine of parent strand is methylated; therefore, repair is only for newly syntehsized strand

in eukaryotes: exact mechanism is not known

what is reverse transcriptase and how does viruses use this?

retroviral enzyme, uses RNA to make DNA (RNA is degraded when done)

creation of ssDNA (cDNA) is then used as the template to make dsDNA

the dsDNA can then be used for transcription of viral genes and to make copies of viral RNA.

Viral dsDNA can be incorporated

into host genome and carried from

mother to daughter cell.

what is didanosine (ddl) and explain its mechanism.

• Converted to dideoxynucleoside triphosphates in cells

• ddI terminates chain growth (elongation) when

• incorporated into DNA by reverse transcriptase.