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CHAPTER 23

PROKARYOTIC REVERSE TRANSCRIPTASES

BERT C. LAMPSON

∗

Department of Health Sciences, East Tennessee State University, Johnson City, TN, USA

∗

lampson@mail.etsu.edu

1. INTRODUCTION

The reverse transcription of the genetic code of an RNA molecule back into a

complementary DNA copy (cDNA) is usually accomplished by a specially dedicated

RNA-dependent DNA polymerase called a reverse transcriptase (RT). Reverse

transcription is presumed to have been a central event in the transition from the

theorized ancient “RNA world” of life, in which RNA molecules served as both

the source of genetic information, as well as, catalytic functions for cellular life,

to the present day DNA-RNA-protein world. Even today reverse transcription

continues to occur in most organisms from human cells to bacteria. For example,

a wide assortment of genetic elements found in plant, animal, and microbial cells use

reverse transcription for at least part of their replication or mobility. These include

RNA viruses, DNA viruses, transposons, introns, and mitochondrial plasmids

(Eickbush, 1994; Eickbush and Malik, 2002). More astonishing than the wide variety

of retroelements that encode a RT is the colossal number of repetitive retrose-

quences, such as the Alu sequences, that have been generated by these elements

in many eukaryotic genomes. These retrosequences are DNAs that usually do not

code for RT but have clearly been produced by reverse transcription of an RNA

molecule (Kazazian, 2004). In addition to the production of these “parasitic” DNAs,

reverse transcription also serves a vital function for most eukaryotic organisms.

Here the essential enzyme telomerase, which functions to maintain the telomere

ends of chromosomes, is a type of RT (Lue, 2004).

Most RTs are placed in two very broad categories based on the phylogenetic

analysis of their amino acid sequence and the type of retroelement that codes for

these polymerases. The first group is found in eukaryotic cells and is usually called

LTR-containing retroelements because their DNA is flanked by long terminal repeat

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404 LAMPSON

sequences. These elements include retroviruses and the virus-like retrotransposons

called Ty in yeast cells. The second group is called non-LTR retroelements because

they are not flanked by long terminal repeats and are a very diverse collection

of elements found in both eukaryotic and prokaryotic organisms. These include

the mobile group II introns, the L1 retrotransposons, and the various retroelements

found in bacteria. Telomerase is phylogenetically related to the RTs from this group

and is thus also included in this second category (Eickbush and Malik, 2002).

Among the best characterized RTs are the polymerases from retroviruses and

include the detailed crystal structure of the RT from the HIV-1 virus (Steitz, 1999).

The focus of this chapter is the recently discovered RTs found in bacteria and the

interesting genetic elements that encode them. These prokaryotic RTs are considered

to be the ancestors of all retroelements found in eukaryotic genomes based on

phylogenetic comparisons (Toor et al., 2001). In addition, the bacterial RTs are

a very diverse group of proteins with a number of novel properties. Thus, these

bacterial RTs represent an emerging new resource for many potential nucleic acid

based technologies like RT-PCR.

Prokaryotic RTs generally fall into one of three different types depending on the

type of retroelement DNA that codes for these proteins. These groups or types are

also in agreement with phylogenetic groupings for RTs determined by comparing

their amino acid sequences. The three types of retroelements are (1) the group II

introns found in both eubacterial and archaeal genomes, (2) retrons, which are also

found in eubacteria and some archaea, and (3) the diversity generating retroelements

that have thus far been found in the eubacteria.

2. GROUP II INTRONS

Among the best understood retroelements of prokaryotic organisms are the group II

introns. The group II introns were initially discovered in the genomes of organelles

like chloroplasts and the mitochondria of yeast (Michel and Lang, 1985). Using

degenerate primers and a PCR screening method, group II introns were subsequently

discovered in a variety of different bacteria as well (Ferat and Michel, 1993).

Like any intron DNA, group II introns interrupt a gene (although this occurs

only occasionally in bacteria) and must be spliced out of the corresponding RNA

transcript to yield a mature RNA encoding a functional gene. In the initial RNA

transcript, independent of any host cell splicing factor, group II introns form an

autocatalytic RNA that can self-splice from the RNA molecule. The excised intron

RNA forms a characteristic lariat structure by a mechanism similar to the splicing

of nuclear introns by the eukaryotic spliceosome (Saldanha et al., 1993).

In addition to self-splicing, most group II intron DNAs found in bacteria contain

an open reading frame (ORF). The product of this ORF gene is a multifunctional

protein with (1) maturase activity that aids in intron splicing (Matsurra et al., 2001),

(2) RT activity that converts the spliced intron RNA into a cDNA copy and, in some

cases (3) a DNA endonuclease that helps the intron insert into a new site in a DNA

molecule (Martinez-Abarca and Toro, 2000; Zimmerly et al., 2001). This intron

PROKARYOTIC REVERSE TRANSCRIPTASES 405

encoded protein allows some intron DNAs to act as retrotransposable elements that

can insert into new DNA molecules (Cousineau et al., 1998, 2000).

2.1. Intron RNA

At 2 to 2.5 kilobase pairs in size, group II intron DNA from bacteria codes for two

important molecules required for intron mobility and splicing. First is the intron

RNA, which will fold into a ribozyme required for self-splicing. And second is

the encoded protein, which aids in splicing and is also required for retrotranspo-

sition (Fig. 1).

Group II intron RNA must fold into a series of complex secondary stem-loop

structures in order to form the autocatalytic ribozyme required for self-splicing.

These stem-loop structures are designated domain I through VI (Fig. 1). The

catalytic core of the intron ribozyme appears to be centered around stem-loop

domain V, which is also the most conserved in its primary nucleotide sequence

among the many different intron RNAs (Toor et al., 2001; Lambowitz and

Zimmerly, 2004). A small stem-loop structure within domain IV appears to function

as a high affinity binding site for the intron RT protein. Here the intron protein binds

to its own intron RNA and helps ensure that the intron RNA folds efficiently into

a catalytically functional ribozyme (Singh et al., 2002). Finally, domain structure

VI contains the so called “bulging A” (large A in Fig. 1) base. This is the internal

adenine nucleotide that will form the 2’–5’ branch linkage with the 5’ terminal base

of the spliced intron to form the lariat intermediate RNA.

Although the basic architecture of the intron RNA described above is shared by all

group II introns, there is a great deal of structural variability especially among the

bacterial introns. There are currently five recognized subgroups or classes of group II

introns found in bacteria (bacterial class A–E) based on the high degree of variation

in their folded RNA structure (Toor et al., 2001; Lambowitz and Zimmerly, 2004).

2.2. Intron Protein

Unlike the group II introns found in organelle genomes, almost all group II introns

found in bacteria have an ORF capable of producing an intron protein, anywhere

from about 415 to 600 amino acids in size. This intron protein is a multifunctional

protein with several functionally defined regions or domains (Fig. 2). Beginning

at the N-terminus and encompassing most of the protein is a region required for

RT activity. Seven blocks (domains 1-7) of conserved amino acid sequence define

this RT domain and are shared among all other RTs including the retroviral RT

of HIV (Xiong and Eickbush, 1990). In addition, this region also defines the so

called fingers and palm regions of the crystal structure of HIV RT suggesting that

this bacterial RT contains a similar folded structure (Steitz, 1999; Blocker et al.,

2005). An additional block of conserved amino acids that is shared among the

bacterial intron RTs and other RTs from non-LTR type transposons, is designated 0

(Blocker et al., 2005).

406 LAMPSON

Figure 1. Group II introns in bacteria are mobile DNAs. From a mRNA transcript of the group II intron,

two important molecules are derived that are required for mobility. First, an intron ORF is translated to

yield a protein with RT activity. Second, the mRNA molecule folds into a complex secondary structure

containing six structural domains designated I-IV (in parentheses). This folded RNA molecule (with

help from the intron protein) forms an active ribozyme that can self-splice. Splicing joins the exon

sequences of the mRNA molecule together and removes the intron sequences as a lariat molecule with

the 5’-end of the intron RNA linked to the 2’ position of an internal adenine nucleotide (large A). The

intron protein binds to the spliced intron RNA to form a ribonucleoprotein complex that will mobilize

the element to new target sites

PROKARYOTIC REVERSE TRANSCRIPTASES 407

Figure 2. The group II intron protein is a multi-functional enzyme. Functional regions of the intron

protein are shown as boxes in the diagram. For example, at the N-terminal half of the protein (left half of

the long open rectangle) is the RT domain defined as seven blocks of conserved amino acids (numbered

boxes 1-7) shared by all RTs. Some highly conserved amino acids in this domain found among group II

intron proteins are shown (single-letter abbreviations above the numbered boxes). Structural modeling

and protease cleavage analysis indicates that the RT domain resembles the fingers-palm folded region

in the crystal structure of HIV RT (brackets in diagram). Just beyond the RT domain is a functional

domain designated “X” that is associated with maturase activity. Evidence indicates that this region

folds into a structure resembling the thumb region in the crystal structure of HIV RT. At the C-terminus

of the intron are two domains designated D and En. Domain D appears to function in binding DNA and

domain En is associated with an endonuclease activity that cleaves DNA

C-terminal to the RT region of the intron protein is a block of amino acids

forming a domain called X. This sequence of amino acids is conserved among group

II intron proteins but is not found in other RT proteins (Zimmerly et al., 2001).

In addition, mutational changes in this region of the protein affect the activity of

intron RNA splicing (Mohr et al., 1993). For this reason, domain X is considered to

be the region of the protein involved in “maturase” activity. For maturase activity,

the intron protein functions as a kind of protein splicing factor. Here, domain X,

and parts of the RT region of the intron protein bind to specific regions of the

intron RNA and help to fold the intron RNA into a stable ribozyme active site

for self-splicing. Although domain X appears to be unique to the group II intron

proteins, it nevertheless contains structural features similar to the thumb region in

the crystal structure of HIV RT. This includes three -helices predicted to form

in the X domain of group II intron proteins (Blocker et al., 2005), and potentially

correspond to the 3 -helices (-H, -I, and -J) in the thumb of HIV RT.

At the C-terminus of the intron protein are two functional domains designated

D and En (Fig. 2). Both of these regions appear to function in different steps that

occur during retrotransposition since truncation of the C-terminal end of the protein to

remove these domains does not affect splicing of the intron RNA (Guo et al., 1997).

The D domain appears to have a DNA binding function that binds to and unwinds

DNA at the site of transposition. The terminal En domain resembles the amino acid

408 LAMPSON

sequence of the H-N-H type DNA endonucleases. This is a member of the so called

homing endonucleases that promote the mobility of intron DNA, of both group I and

group II introns, by recognizing and cleaving DNA at intron homing sites (see intron

mobility below). There are several different types of these endonucleases such as the

H-N-H family. The H-N-H family of enzymes contain conserved histidine amino

acids flanking an asparagine, and include colicins, phage encoded endonucleases and

the intron homing enzymes (Belfort and Roberts, 1997). This endonuclease activity

carries out the second-strand cleavage event during retro-mobility (Guo et al., 1997).

Many of the group II intron proteins from bacteria do not contain the En domain and

thus carry out retro-mobility without this endonuclease activity by a slightly different

mechanism (Ichiyanagi et al., 2002; Zhong and Lambowitz, 2003).

2.3. Reverse Splicing and Intron Mobility

As mentioned above, group II intron RNA acts as a self-splicing ribozyme to excise

itself from mRNA. The splicing reaction can also occur in the reverse direction with

the excised intron RNA re-inserting back into the mRNA molecule in vitro (Morl

and Schmelzer, 1990). This ability to reverse splice turns out to be a key event in

the mobility of group II introns into new locations, except in this case the intron

RNA reverse splices directly into a DNA molecule rather than an RNA molecule.

Mobility of group II introns in bacteria is divided into two types of insertion

events. The first is a highly efficient, site-specific insertion of the intron into what

is called the homing site for the intron. The homing site is simply an analogous

DNA sequence lacking the intron element. This type of mobility event is called

retrohoming and does not involve homologous recombination (Cousineau et al.,

1998). In rare cases, apparently, a group II intron may insert into an unrelated

or non-allelic DNA site in a process called retrotransposition (Cousineau et al.,

2000). In both cases reverse splicing and reverse transcription are key events in the

mobilization of the intron to a new site.

From the limited information derived from sequenced bacterial genomes, group

II introns appear to be the most common of the RT encoding elements found in

the prokaryotes. About one fourth of all the bacterial genomes sequenced appear

to contain at least one group II intron. Group II introns appear rarely among the

few archaeal genomes that have been sequenced. An analysis of the insertion sites

of group II introns in bacteria indicates that these intron DNAs only occasionally

interrupt an ORF that requires the subsequent splicing of the intron to produce

a functional gene product (Dai and Zimmerly, 2003). More commonly, group II

introns are found associated with some kind of mobile DNA such as plasmids, IS

elements, or transposons. When they are found in the chromosome, they commonly

insert into intergenic DNA between two intact genes (Dai and Zimmerly, 2003).

Although the group II introns appear to be fairly common in prokaryotic genomes,

they were not the first type of RT containing element to be discovered in bacteria.

That distinction goes to the next type of retroelement found in the prokaryotes, the

retron elements.

PROKARYOTIC REVERSE TRANSCRIPTASES 409

3. RETRONS AND msDNA

These strange retroelements, whose function is essentially unknown, have been

observed to do only one thing; they synthesize large quantities of multicopy single-

stranded DNA (msDNA). Indeed, it was the discovery of this unusual satellite

msDNA that led to the first discovery of RT from a prokaryotic organism (Lampson

et al., 1989b; Lim and Maas, 1989).

3.1. Organization of Retron DNA

Retrons are distinct DNA sequences of about 2000 base pairs that are found inserted

into the bacterial chromosome or as part of a prophage DNA element. The functional

organization of the retron is based on the only known activity of these retroelements,

that is, the production of msDNA. Other possible functions like transposition are

not known.

All retron elements have at least one large ORF designated ret (Fig. 3). The

amino acid sequence of this ORF product, which can range from 298 to 586 amino

acids, reveals an easily recognizable RT that is similar to eukaryotic RTs (Lampson

et al., 1989b, 2002). Experiments with mutational variants, containing a deleted

or inactivated ORF showed that these retrons fail to produce msDNA (Lampson

et al., 1989b). Thus, the synthesis of msDNA requires a functional RT and is the

first example of a reverse transcribed cDNA in bacteria. Immediately upstream to

the start of the ORF are two genes designated msr and msd. These two genes are

also required to synthesize msDNA and are situated in opposite directions such

that their 3’ends overlap by several bases (Fig. 3 and Fig. 5). The genes msr,

msd and ret are transcribed together as one mRNA and appear to be under the

control of a promoter found within the retron DNA just upstream of msr (Herzer

et al., 1992). Because msDNA is not an autonomously replicating satellite DNA,

the three genes msr, msd, and ret essentially form an operon to synthesize msDNA.

Some retrons may code for a second ORF but its function remains unknown

(Sun et al., 1991).

3.2. The Retron RT

As noted for the intron protein of bacterial group II introns, the retron RT also

contains the seven conserved domains 1-7 that roughly correspond to the fingers

and palm region in the crystal structure of HIV RT (Fig. 3). Also like the intron

protein, the retron RT contains the spacer regions 2a, 3a, and a very small 4a. The

function of these spacer regions is not well understood. Beyond the RT domain at the

C-terminus of the retron RT is a unique region of amino acids not found in

other RTs, and sometimes designated domain “Y” (Fig. 3). The circular dichroism

spectrum of this region in retron RT-Ec86 indicates mostly alpha-helical struc-

tures. Thus, this C-terminal domain may correspond to the three alpha-helicies

(-H, -I, and -J) of the thumb region in the crystal structure of HIV RT

410 LAMPSON

Figure 3. Retron DNA: an operon for making msDNA. Retron elements, composed of about 2 Kb of

unique DNA (thin line), are usually inserted into the chromosome (black boxes) or into prophage DNA.

Most retron elements contain a single ORF encoding RT (ret). Adjacent to ret are two genes designated

msr and msd. The msr sequence corresponds to the RNA molecule in msDNA and the msd sequence

corresponds to the DNA chain in msDNA. A cDNA copy of the msd gene produced by the retron RT

leads to production of the satellite DNA known as msDNA. Like all RTs, the retron protein (bottom

diagram) contains the seven blocks of conserved amino acids (boxes labelled 1-7) that fold into the

fingers-palm region that is observed in the crystal structure of HIV RT (labelled brackets at the bottom).

Some of the more highly conserved amino acids in this region of the retron RT are shown as single-letter

abbreviations above the boxes. Based on circular dichroism analysis, the C-terminal part of the retron

protein appears to have a structure similar to the thumb region in the crystal structure of HIV RT. This

region of the protein binds to a specific structure in the template-primer RNA used to make msDNA.

Some retron RTs have an extended N-terminal region of unknown function (shaded rectangle)

PROKARYOTIC REVERSE TRANSCRIPTASES 411

(Yamanaka et al., 2002; Inouye et al., 2004). Experiments with just the purified 66

amino acid C-terminal domain of retron RT-Ec86 indicates that this thumb region

recognizes and binds to a specific stem-loop structure in the RNA template molecule

used to synthesize msDNA. As discussed below, this is a critical step in the unusual

priming mechanism used to initiate the synthesis of msDNA (Inouye et al., 1999,

2004). It is interesting to note the similarity between the C-terminal thumb region

of retron RT and the maturase (domain “X”) of group II intron proteins. In both

proteins this thumb structure appears to recognize and bind to a specific stem-loop

structure in the RNA transcript encoding the RT. In the case of the intron protein

this binding helps to maintain the folded structure of the intron RNA for splicing.

In the case of the retron protein this binding helps to maintain the structure needed

to prime the start of synthesis of msDNA (see below).

Some retron RTs have an extremely long N-terminal extension beyond the RT

domains 1 through 7. For example, the RT from retron Mx162, from the myxobac-

terium Myxococcus xanthus, contains a 165 amino acid extension before the RT

domain 1 (Inouye et al., 1989). The function of this extended region is unknown.

3.3. The Unique Structure of msDNA

msDNA is a strange satellite DNA first discovered in M. xanthus (Yee et al.,

1984). In the case of msDNA-Ne144 from the related myxobacterium Nannocystis

exedens, the satellite DNA is composed of a 144 nucleotide single-stranded DNA

that folds into a stable stem-loop structure. Covalently linked to the 5’ end of the

DNA chain is a 72 base, single-strand of RNA. However, the RNA strand retains

both a free 5’ end and a free 3’ end and is instead joined to the DNA strand at an

internal guanosine nucleotide via a 2’– 5’ phosphodiester bond (circled G, Fig. 4).

msDNA is thus a unique molecule in that a DNA molecule is joined to an RNA

molecule by a 2’–5’ phosphodiester linkage (Dhundale et al., 1987; Furuichi et al.,

1987). In addition to RNA and DNA, msDNA is most likely bound in a complex

with protein. The major protein in this DNA-RNA-protein complex appears to be

the retron RT (Lampson et al., 1990), although other host proteins may also bind

to msDNA. For example, RNase H produced by the host cell may bind to msDNA,

at least initially, since it appears to be involved in the synthesis of msDNA (Lima

and Lim, 1995; Shimamoto et al., 1995).

Different retron elements produce distinct msDNAs with generally little similarity

in the primary nucleotide sequence of either the DNA molecule or the RNA

molecule. All msDNAs, however, appear to contain, at least initially, the basic

secondary structure shown in Fig. 4. Some msDNAs, apparently, undergo some

modification after they are synthesized. For example, the entire RNA strand

including the 2’–5’ branch linkage is removed from msDNA-Ec83 from Escherichia

coli resulting in a mature msDNA that is only composed of a single-stranded DNA

molecule (Lim, 1992; Kim et al., 1997). The unusual structure of msDNA appears

to be a consequence of the way this DNA is synthesized (see below). Why some

msDNAs are then further modified is unknown.