genetics lecture 19 + 20

initiation of DNA synthesis

- elongation of polynucleotide chain by DNA polymerase II requires primer with free 3' - OH

primase

- RNA polymerase

- recruited to replication form by helicase

- synthesizes RNA primer

- provides free 3' - OH required by DNA polymerase III elongation

antiparallel strands

- two strands of double helix antiparallel: 5'-3' and 3'-5'

- DNA polymerase III only synthesizes 5'-3'

continuous DNA synthesis

- only one strand can serve as template for continuous DNA synthesis (leading strand)

discontinuous DNA synthesis

- lagging strand, opposite of DNA template

- lagging strand synthesized as okazaki fragments each with RNA primer

DNA polymerase I

- removes primers on lagging strand

DNA ligase

- catalyzes formation of phosphodiester bonds

- seals nicks and joins fragments

concurrent synthesis

- DNA synthesized on both leading and lagging strands concurrently

- lagging strand looped, inverts physical but not biochemical direction

- DNA clamp prevents core enzyme dissociation from template

proofreading and error correction

- integral part of DNA replication

- DNA polymerase is accurate, but synthesis is not always perfect

- DNA polymerase exonuclease activity of 3'-5' allows for excise of nucleotides

mutations

- interrupt/impair aspects of replication

- lethal mutations/conditional mutations: ligase-deficient mutations, proofreading-deficient mutations, temperature-sensitive mutations

temperature-sensitive mutations

- example of conditional mutation

- may not be expressed at particular permissive temperature

- mutant cells grown at restrictive temperature and mutant phenotype expressed

eukaryotic and bacterial DNA replication

- double-stranded DNA unwound at ORI

- replication forks formed

- bidirectional synthesis creates leading and lagging strands

- eukaryotic polymerases require four deoxyribonucleoside triphosphates, template, and primer

eukaryotic DNA replication

- more complex

- more DNA than prokaryotic cells

- linear chromosomes

- DNA complexed with nucleosomes

eukaryotic replication initiation

- eukaryotic chromosomes contain multiple ORIs

- multiple "replication bubbles" visible under electron microscope

- facilitates rapid synthesis of large quantity of DNA

DNA replication in yeast

- yeast genomes contain 250-400 origins

- yeast ORI: autonomously replicating sequences (ARSs)

- 120 base pairs of consensus sequences (sequence that is the same in all yeast ARSs)

multiple eukaryotic DNA polymerases

DNA polymerases involved in nuclear genome DNA

replication

- human genome encodes for 14 different DNA

polymerases

- only three involved in DNA replication

- polymerases α, δ, and ε: involved in initiation and

elongation

- pol α enzyme: possesses low processivity, RNA primer synthesis during initiation on leading

and lagging strands.

polymerase switching

- occurs once primer is in place

- pol α dissociates from template and ε replaces pol α

for elongation

- pol δ synthesizes lagging strand

- pol ε synthesizes leading strand

replication through chromatin

- eukaryotic DNA is complexed with binding proteins (chromatin)

- 200 base pair nucleosomes wrap around 8 histone proteins (octet)

- nucleosomes must be stripped away before polymerase can begin synthesis

- assembly of nucleosomes into two daughter strands carried away by chromatin assembly factors (CAFs)

telomeres

- long stretches of short repeating sequences preserve the integrity/stability of chromosomes

- eukaryotic chromosomes are linear (DNA "ends" create problems)

- double-stranded "ends" of DNA molecules at terminal linear chromosomes potentially resemble double-stranded breaks (DSBs)

- double-stranded loose ends vulnerable to degradation by nucleases

replication at the telomere

- telomeric sequence in humans: 5'-TTAAGGG-3'

- telomeres protect the ends of linear chromosomes with the use of T-loops and shelterin complex

telomerase

- capable of adding several more repeats to 3' end of G-rich strand

- telomerase RNA component (TERC) serves as both a guide and template for synthesis of DNA complement

- telomerase reverse transcriptase (TERT): catalytic unit of telomerase enzyme

telomeres in disease, aging, and cancer

- several human diseases associated with loss of telomerase activity and short telomeres

- connection between telomere length + aging topic of research and speculation

- cells suffer chromosomal damage when telomeres become short (enter senescence) and cell division ceases

- human cancer cells retain telomerase activity, key to their immortality

organization of DNA

- DNA is organized into genes

- basic units are genetic function are organized into chromosomes

- molecular analysis with light and electron microscopy that has provided insights into chromosome organization (eukaryotic structures, polytene, and lampbrush chromosomes)

viral and bacterial chromosomes

- single nucleic acid molecules

- largely devoid of associated proteins

- much smaller than eukaryotic chromosomes

- contain less genetic information

viral chromosomes

- nucleic acid, either DNA or RNA, single or double stranded

- circular (closed loops) or linear molecules

- ΦX174 bacteriophage is dsDNA

- bacteriophage lambda is linear dsDNA

- circularity is not required for replication of virus

viral genetic material

- inert until released into host cell

- able to package long DNA into small volume just like bacteria and eukaryotic cells

- viruses, bacteria, and eukaryotic cells all have ability to package DNA into small volumes

bacterial chromosomes

- circular, double-stranded DNA compacted into nucleoid

- E. coli most extensively studied, has 1.2mm of chromosome in length

- bacterial DNA associated DNA binding proteins

- HU and H-NS (histone-like nucleoid structuring protein)

- proteins fold and bend DNA creating coils for compaction of DNA

mitochondria and chloroplasts

- contain their own DNA

- inherited through maternal cytoplasm in most organisms

- the nature of their DNA is similar to viruses and bacteria

mitochondrial DNA (mtDNA)

- mtDNA in most eukaryotes exists as double-stranded closed circle

- does not contain chromosomal proteins

- introns mostly absent from mitochondrial genes

- gene repetition seldom present

- replication of mtDNA dependent on enzymes coded by nuclear DNA

chloroplast DNA (cpDNA)

- chloroplast provides photosynthetic function for plants

- cpDNA shares similarities to DNA found in prokaryotes

- circular, double-stranded, and free of associated proteins found in eukaryotic DNA

- larger than mtDNA (larger # of genes)

- has both introns and duplications

polytene chromosomes

- found in various tissues

- can be seen in nuclei of interphase cells

- represent paired homologs

- not usually found in eukaryotic cells

- light microscopy revealed chromomeres (individualized bands)

- DNA of paired homologs undergoes many rounds of replication without strand separation or cytoplasmic division

puff regions

- localized uncoiling during genetic activity

- visible manifestations of high-level gene activity (transcription produces RNA)

lampbrush chromosomes

- meiotic chromosomes first studied in oocytes of sharks

- large with extensive DNA looping

- found in most vertebrate oocytes and spermatocytes of some insects

- easily isolated from oocytes in diplotene stage of prophase I of meiosis

- contain large number of condensed areas (chromomerse)

chromatin

- at interphase: eukaryotic chromosomes uncoil and decondense into chromatin

- during interphase: chromatin is dispersed throughout nucleus and replicated

- during cell division: chromatin coils and condenses back into visivle chromosomes

histones

- eukaryotic chromatin associated with histones: positively charged proteins associated with chromosomal DNA in eukaryotes + less positively charged nonhistones

- histones play essential role in structure of proteins associated with DNA

- 5 main types: all contain positively charged lysine and arginine, allows for electrostatic bondings to phosphates

chromatin structure

- digestion of chromatin by endonucleases

- chromatin fibers are composed of linear array of spherical particlaes called nucleosomes

- histones H2A, H2B, H3, and H4 occur as tetramers

- nucleosome core particle created when nuclease digestion is extended and 200bps of DNA is removed

chromatin remodeling

- important topic in epigenetics

- DNA is inaccessible to interactions due to chromatin fiber complexed with histones

- to accommodate DNA (protein interactions, replication, gene expression), chromatin structure must relax compact structure + have reversal mechanism for inactivity

chromatin importance

- x-ray diffractions studies revealed twists and turns of DNA superhelix encircled with histones

- nucleosome: principal packaging unit of DNA in eukaryotic nucleus

- unstructured histone tails are not packed into folded histone domains within nucleosome

- tails of H4 make connections with adjacent nucleosomes

- tails devoid of secondary structure protrude through minor groove