DNA Damage

Figure 5-46. A summary of spontaneous alterations likely to require DNA repair.

The sites on each nucleotide that are known to be modified by spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows), and uncontrolled methylation

by the methyl group donor S-adenosylmethionine

(green arrows) are shown, with the width of each arrow indicating the relative

frequency of each event. (After T. Lindahl, Nature 362:709–715, 1993. ©

Macmillan Magazines Ltd.)

Deamination

of Cytosine

Thyamine

dimers

Nucleases•

Cleave nucleotide sequences

•

DNases

and RNases

and non specific nucleases

•

ss

and ds

specificity•

Exonucleases

(remove nucleotide from the

end)•

Endonucleases

(recognize palindromic

ds

DNA sequences)

Restriction endonucleases

•

Three types (I, II, and III) –

I and III require ATP

•

Type II are used as common molecular biology tools

Type II restriction enzymes

•

Recognize and cleave particular sequencesFor example, BamHIGGATCC

5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’

BamHI

5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’

5’-N-N-N-N-G G-A-T-C-C-N-N-N-N-3’3’-N-N-N-N-C-C-T-A-G G-N-N-N-N-5’

“sticky ends”

–

overhanging sequence

Why do bacteria have endonucleases?

How do they avoid digesting their own DNA?

Overview•

DNA structure –

A, B, and Z DNA

•

DNA intercelators

and groove binders•

Thermal melting of DNA

•

DNA tertiary structure•

DNA methylation

•

DNA damage•

nucleases

Which of the following statements correctly describes B-DNA

A.

B-DNA is usually found in solutions of reduced water

B.

B-DNA displays a wider helix in comparison to Z-DNA

C.

B-DNA forms a grooved left-handed helixD.

B-DNA has a helix shorter and wider than A-DNA

RNA

Arrangement in three dimensions

Beyond the Four Bases

Elements of RNA Secondary Structure

Elements of RNA Tertiary Structure

Messenger RNA (mRNA)Transcription product of DNA

•

In prokaryotes, a single mRNA contains the information for synthesis of many proteins

•

In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns

and exons

Eukaryotic mRNA 5’

Cap

A phosphate is released by hydrolysis. The diphosphate

5′

end then attacks the α-phosphorus atom of GTP to form a very unusual 5′-5′

triphosphate

linkage. This distinctive terminus is called a cap .

The N-7 nitrogen of the terminal guanine is then methylated

by S-adenosylmethionine

to form cap 0. The adjacent riboses

may be methylated

to form cap 1 or cap 2.

Caps contribute to the stability of mRNAs by protecting their 5′

ends from phosphatases

and nucleases. In addition, caps enhance the translation of mRNA by eukaryotic proteinsynthesizing

systems

Eukaryotic mRNA poly A tail

mRNA molecule devoid of a poly(A) tail is usually a much less effective template for protein synthesis than is one with a poly(A) tail. –

enhances translation of mRNAThe half-life of an mRNA molecule may also be determined in part

by the rate of degradation of its poly(A) tail. –

enhances stability of mRNA

Transfer RNA (tRNA)

•

Recruits amino acid to the ribosome to synthesize protein

•

Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem

•

Many non-canonical base pairs found in tRNA

Secondary Structure of tRNA

R = amino acid

Tertiary Structure of tRNA

5’3’

Noncanonical

base pairing and unusual bases in tRNA

Ribosomal RNA•

Facilitate protein synthesis

•

Ribosomes

are about 2/3 RNA, 1/3 protein •

rRNA

serves as a scaffold for ribosomal proteins

•

23S rRNA

in E. coli is the peptidyl

transferase

– catalytic! RIBOZYME

Secondary structure of rRNA

Small nuclear RNA (snRNA)

snRNP Size of snRNA(nucleotides)

Role

U1 165 Binds the 5′

splice site and then the 3′

splice siteU2 185 Binds the branch site and forms part of the

catalytic centerU5 116 Binds the 5′

splice siteU4 145 Masks the catalytic activity of U6U6 106 Catalyzes splicing

Participate in splicing the hnRNA

to form the mature mRNA

These RNA molecules and proteins assemble to form the Splicesome

snRNA

catalysis

Hammerhead –

catalytic RNASelf cleaving RNA involved in replication of single stranded viroid

(RNA infectious agents of plant disease)

Small interfering RNA (siRNA)•

Variety of roles in biology –

most characterized is the interference of the expression (translation) of a specific gene

RISC -

RNA-induced silencing complexes

DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not?•

Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis

•

DNA, lacking 2'-OH is more stable •

This makes sense -

the genetic material

must be more stable •

RNA is designed to be used and then broken down

Hydrolysis of Nucleic Acids

•

RNA is resistant to dilute acid •

DNA is depurinated

by dilute acid

•

DNA is not susceptible to base •

RNA is hydrolyzed by dilute base

RNA World

Chemical view: Abiotic

nucleotide chemistry → RNA-catalyzed biochemistry.

Biochemical view:RNA-based life → Protein/DNA-based life.

Why RNA vs

Peptide or DNA

How could protein synthesis work without protein and DNA?

•

Polypeptides would have played only a limited role early in the evolution of life because their structures are not suited to self-replication in the way that nucleic acid structures are. However, polypeptides could have been included in evolutionary processes indirectly. For example, if the properties of a particular polypeptide favored the survival and replication of a class of RNA molecules, then these

RNA molecules could have evolved ribozyme

activities that promoted the synthesis of that polypeptide. This

method of producing polypeptides with specific amino acid sequences has several limitations. First, it seems likely that only relatively short specific polypeptides could have been produced in this manner. Second, it would have been difficult to accurately link the particular amino acids in the polypeptide in a reproducible manner. Finally, a different ribozyme

would have been required for each polypeptide. A critical point in evolution was reached when an apparatus for polypeptide synthesis developed that allowed the sequence of bases in an RNA molecule to directly dictate the sequence of amino acids in a polypeptide. A code evolved that established a relation between a specific sequence of three bases in RNA and an amino acid. We now call this set of three-base combinations, each encoding an amino acid, the genetic code. A decoding, or translation, system exists today as the ribosome and associated factors that are responsible for essentially all polypeptide synthesis from RNA templates in modern organisms. The essence of this mode of polypeptide synthesis is illustrated in Figure 2.8.

•

An RNA molecule (messenger RNA, or mRNA), containing in its base sequence the information that specifies a particular protein, acts as a template to direct the synthesis of the polypeptide. Each amino acid is brought to the template attached to an adapter molecule specific to that amino acid. These adapters are specialized RNA molecules (called transfer RNAs or tRNAs). After initiation of the polypeptide chain, a tRNA

molecule with its associated amino acid binds to the template through specific Watson-Crick base-pairing interactions. Two such molecules bind to the ribosome and peptide-bond formation is catalyzed by an RNA component (called ribosomal RNA or rRNA) of the ribosome. The first RNA departs (with neither the polypeptide chain nor an amino acid attached) and another tRNA

with its associated amino acid bonds to the ribosome. The growing polypeptide chain is transferred to this newly bound amino acid with the formation

of a new peptide bond. This cycle then repeats itself. This scheme allows the sequence of the RNA template to encode the sequence of the polypeptide and thereby makes possible the production of long polypeptides with specified sequences. The mechanism of protein synthesis will be discussed in Chapter 29. Importantly, the ribosome is composed largely of RNA and is a highly sophisticated ribozyme, suggesting that it might be a surviving relic of the RNA world.