Macromolecular Genetics of Life

Cell Biology, BIOL310

Advancement of eukaryotic cell function and activities governed by three “R”s

• “Ability of cells to maintain a higher degree of order in a chaotic universe depends upon the accurate duplication of vast quantities of genetic information”

• Maintaining this requires: – Replication with efficiency

(speed times accuracy) – Repair of mistakes and

damages that would ultimately lead to dysfunctional protein

– Recombination allows for the generation of functional diversity to better adapt, survive, and thrive in an evolving, changing world

Genetic stability via maintenance of DNA sequences is necessary for survival

• Germ cells transmit genetic information from parent to offspring

• Somatic cells are cells that give rise to specialized tissue and cells in the body – each differentiated to carry

out instructed tasks – Division of labor based on

their ability to access the genetic code

Synthesis of the polymers for protein, carbohydrates, and nucleotides

harvests the power of water

Base-pairing underlies DNA replication and repair and highlights

the value of protein-DNA interactions • Process of recognizing a

nucleotide within a single strand template DNA and inserting it’s complimentary base (or nucleotide) – This is principally

mediated by the H-bond interactions between A-T and G-C

Semi-conservative inheritance highlights the importance of covalent bonds vs. H-

bond in control of information

As all life follows the rule of semi-conservative inheritance, all cellular life is

descendent from a predecessor cell • Each of the two daughter

cells inherits a new DNA double helix containing one original and one new strand – semi-conservative

• In all rounds of replication, each of the two DNA strands are used as templates to form complementary DNA strands

Eukaryotes DNA replication takes part only during S phase of cell cycle

• Replication occurs only during DNA synthesis (S) phase of the cell cycle

• Typically last about 8 hours in mammalian cells – About 40 minutes for

single celled organisms like yeast

• Directly proportional to size and number of start sites

DNA synthesis begins at replication origins, which are conserved sequences to which proteins bind in order to identify start site

• DNA replication is begun by initiator proteins that bind to dsDNA at specific, conserved sequences – These initiator proteins

bind and pry the two strands apart, breaking the H-bonds between bases

• The conserved sequences, or positions, are called replication origins

Eukaryotic chromosomes contain multiple origins of replication

• Because of the vast size of the eukaryotic genome, multiple origins of replication must exist to which DNA replication of numerous parts can proceed concomitantly – Speed x Accuracy – Within in a replication unit,

individual origins are spaced at intervals of about 30L-250K nucleotides apart from one another

Different regions on the same chromosome replicate at distinct times

• Different regions of each chromosome are replicated in a reproducible order during S phase – This can be identified by the

multiple clusters of replication forks seen at different time points by radiography labeling

• Highly condensed chromatin replicated late, while genes in less condensed chromatin is replicated early – Accessibility is governed by

differential temporal expression of genes

Synthesis is catalyzed by DNA polymerase catalysis of nucleotide addition of nucleotides in a copy-paste

The backbone of DNA is assembled through simple monomer addition to a growing chain – a theme of Cell Biology • Multi-enzyme complex that

contains DNA polymerase synthesizes the DNA of both new daughter strands.

• Importantly, DNA is always synthesized in the 5’ 3’ direction – It is never, ever possible to

synthesize in the 3’ 5’ direction

• Thus, a mechanism must be put into place to add DNA in the 5’3’ direction while simultaneously reading 5’ 3’

H + OH

Finding a work around to traveling and writing in the 5’ 3’ direction, the

cell uses RNA primers-Okazaki Fragm. • DNA synthesis on the lagging

strand must be made initially as a series of short DNA molecules – These molecules are called

okazaki fragments • For the lagging strand, the

direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth – Most importantly, it is still made

in the 5’3’ direction while reading also in the 5’3’ direction in a discontinuous fashion

Finding a work around to traveling and writing in the 5’ 3’ direction, the cell uses RNA primers-Okazaki Fragments

• Most importantly, it is still made in the 5’3’ direction while reading also in the 3’5’ direction in a discontinuous fashion

• The DNA polymerase knows where to start and stop thanks to a START SEQUENCE called a RNA primer

DNA primase synthesizes RNA primer molecules for lagging strand synthesis

• A RNA primer is made downstream

• The short RNA primer can be elongated by DNA polymerase to make the complimentary DNA strand – It begins by generating the

okazaki fragment that runs until it meets up with the 5’ end of the RNA primer of the previous fragment.

• DNA repair machinery remove the RNA primers and replace them with DNA nucleotides,

• DNA ligase effectively joins the 3’ end of one okazaki fragment with the 5’ end of the next okazaki fragment to seal the DNA backbone

The drawback to using RNA primers for lagging strand and working backwards is

that eventually you run out of room • Remember, the lagging

strand needs RNA primers to place downstream so it can synthesize back in the correct 5’3’ fashion while reading in the 3’5’

• A problem then occurs when you reach the very end of the DNA in (non-circular) Eukaryotic genomes – How do you then add

primers to synthesize back when there is no more room?

Telomerase replicates chromosome ends

• Eukaryotic cells have evolved specific sequences at the ends of their chromosomes – This is generally a rich repeat of

GGGTTA sequences – sequences serve as DNA binding motif

• Secondly, the eukaryotic cells use telomerase enzymes that extends the 3’ end by adding an RNA-sequence template at the ends of chromosomes – Now, DNA polymerase has more

template to work with to synthesize the end of the DNA

Telomere length is regulated by cells, deregulation lead to uncontrolled

division/cell replication • The process of telomere

extension and contraction is variable – thus lengths of telomere ends

can\are different from cell to cell

• In effect, the absence of telomerase granted RNA primers means the end of appropriate DNA replication and senescence of that cell – I.e. No more daughter cells;

cellular division is lost • Thus the replication extent of

life can be modulated by telomerase activity

Numerous different proteins cooperate in a concerted effort to

drive DNA replication

Proteins at a replication fork cooperate to form a replication machine

Replication requires work, and the cell readily uses it’s ATP molecules to drive

unfavorable energetic reactions

DNA replication requires several proofreading mechanisms to ensure

the integrity of the copy • DNA thus requires the

necessity to ensure that the copies it makes are not filled with errors – or every subsequent copy will be faulty. – DNA is so efficient that

there is only 1 mistake for every 109 nucleotides copied

• The mistakes occur at the level of mismatched base-pairing

Energetically favorable nucleotides provide means to ensure correct addition

• DNA polymerase performs the first proofreading step immediately before a new nucleotide is added to the growing chain – The correct nucleotide has a

higher affinity for a moving polymerase that does the incorrect nucleotide

• Wrong pairing is energetically unfavorable – Thus the first true

proofreading step is to ensure energetically favorable nucleotides

Strand-directed mismatch repair system removes replication errors

• Strand-directed mismatch repair system detects the potential for ‘distortion’ in the DNA helix from the misfit between non-complementary bases

• After removing the DNA from the newly synthesized strand, the DNA repair system adds in the appropriate nucleotides following the original DNA template – How does the machinery tell

the template strand vs. the newly synthesized strand?

The rate of mutation is exactly related to the number of proteins (i.e. genes)

• Mutation rates: rate at which changes in nucleotide base pairs occur; often times equated to observable changes (phenotype)

• For an organism to maximize productivity, it has evolved to a saturated point balancing rate of mutations versus the probability of accruing that mutation in genes – This has put pressure on the

genome to keep only a maximum of 50K genes