Recombinant Saccharomyces cerevisiae Inosine triphosphate pyrophosphatase (HAM1)

What is HAM1 inosine triphosphate pyrophosphatase in Saccharomyces cerevisiae?

HAM1 is a 197-amino acid protein that functions as an inosine triphosphate pyrophosphatase in Saccharomyces cerevisiae. The protein plays a crucial role in nucleotide metabolism by hydrolyzing potentially mutagenic non-canonical nucleotides. The HAM1 gene was initially identified through studies of yeast mutants that exhibited sensitivity to the mutagenic base analog 6-N-hydroxylaminopurine (HAP) . The protein's function appears to be conserved across species, with homologs found in various organisms including humans (ITPA).

When studying HAM1, researchers should consider its primary sequence (including the N-terminal methionine): MSNNEIVFVTGNANKLKEVQSILTQEVDNNNKTIHLINEALDLEELQDTDLNAIALAKGKQAVAALGKGKPVFVEDTALRFDEFNGLPGAYIKWFLKSMGLEKIVKMLEPFENKNAEAVTTICFADSRGEYHFFQGITRGKIVPSRGPTTFGWDSIFEPFDSHGLTYAEMSKDAKNAISHRGKAFAQFKELYQNDF . This sequence information is essential for designing expression constructs, developing purification strategies, and conducting structural analyses.

What is the genetic organization of the HAM1 gene in S. cerevisiae?

The HAM1 gene in S. cerevisiae is located on the right arm of chromosome X, positioned between the cell division cycle genes cdc8 and cdc11 . Genetic mapping and sequencing studies have revealed that the HAM1 locus contains a 3.4 kb functional fragment with three open reading frames (ORFs), one of which (coding for the 197 amino acid protein) represents the HAM1 gene .

In experimental approaches, researchers can use this positional information for targeted genetic manipulations. For example, LEU2+ disruptions of the HAM1 gene's promoter and N-terminal regions have been constructed, resulting in moderate and strong sensitivity to HAP, respectively . These disruption strains are valuable tools for investigating HAM1 function through complementation studies and phenotypic analyses.

How does HAM1 protect cells against nucleotide analogs?

HAM1 protects cells by hydrolyzing mutagenic nucleotide analogs before they can be incorporated into DNA or RNA. This is demonstrated by the increased sensitivity of ham1 mutants to 6-N-hydroxylaminopurine (HAP), a base analog with mutagenic and lethal effects . The protective mechanism involves:

• Recognition of non-canonical nucleotides by HAM1

• Hydrolysis of the triphosphate bond

• Generation of monophosphate forms that cannot be incorporated into nucleic acids

To study this protective function experimentally, researchers can expose wild-type and ham1 mutant yeast to varying concentrations of HAP and measure survival rates and mutation frequencies. Complementation studies using cloned HAM1 genes can confirm that the protective effect is directly attributable to HAM1 function .

What are the key structural features of HAM1 protein?

The HAM1 protein consists of 197 amino acids with several conserved domains that are essential for its enzymatic activity . While the complete three-dimensional structure details weren't provided in the search results, protein analysis reveals a tertiary structure likely containing nucleotide-binding pockets typical of pyrophosphatases.

Key structural features include:

For experimental studies of HAM1 structure, researchers should consider expression systems that maintain proper protein folding. The yeast expression system is particularly advantageous as it "integrates the advantages of the mammalian cell expression system" while being more economical and efficient . This system allows for post-translational modifications such as glycosylation, acylation, and phosphorylation that ensure native protein conformation.

What are optimal expression systems for recombinant HAM1 production?

Based on the available data, the yeast expression system appears optimal for HAM1 production. This system offers several advantages for recombinant HAM1 production:

• It is "the most economical and efficient eukaryotic system for secretion and intracellular expression" .

• It allows for post-translational modifications similar to those in mammalian cells.

• It produces protein that is "very close to the natural protein" .

• It achieves higher yields compared to mammalian expression systems.

The recombinant HAM1 protein expressed in yeast can be tagged with a His tag for purification purposes, achieving purity levels greater than 90% . This approach is suitable for various applications, including ELISA and enzymatic activity assays.

For researchers considering alternative expression systems, it's worth noting that E. coli, mammalian cells, and baculovirus infection systems are also viable options, though they may differ in "price and lead time" .

How does HAM1 activity relate to genome stability and mutagenesis?

For researchers investigating the relationship between HAM1 and genome stability, several methodological approaches are valuable:

• Mutation rate analysis: Measure spontaneous and induced mutation rates in wild-type and ham1 mutant strains.

• DNA damage response studies: Analyze interactions between HAM1 and DNA repair pathways.

• Genome-wide sequencing: Compare mutation spectra in wild-type and ham1 strains exposed to mutagenic agents.

A comprehensive experimental design might include:

These experiments can reveal the extent to which HAM1 contributes to genome stability under various conditions and in response to different mutagenic agents.

How does the genomic context affect HAM1 function?

The genomic location of HAM1 on chromosome X between cdc8 and cdc11 may influence its expression and function . While the search results don't provide specific information about how this genomic context affects HAM1, research in related fields suggests potential implications.

For instance, in yeast, the meiotic chromosome axis influences recombination and DNA double-strand break (DSB) formation. Proteins like Hop1 are important for DSB formation, and their levels "which vary along the lengths of chromosomes, are positively correlated with double-strand break levels" . By analogy, the genomic context of HAM1 might influence its expression patterns during different cellular states.

Researchers investigating genomic context effects could employ:

• Chromosome engineering: Relocating the HAM1 gene to different genomic locations and measuring expression and function.

• ChIP-seq analysis: Mapping chromatin modifications and protein binding at the HAM1 locus.

• Transcriptome analysis: Comparing HAM1 expression patterns with neighboring genes under various conditions.

These approaches could reveal how the genomic neighborhood influences HAM1 regulation and function, potentially uncovering new regulatory mechanisms.

How might HAM1 research contribute to biotechnology applications?

HAM1 research has several potential biotechnology applications:

• Enzyme-based biosensors: Utilizing HAM1's specificity for non-canonical nucleotides to develop detection systems for mutagenic compounds.

• Improved protein expression systems: The understanding that HAM1 protects against nucleotide analogs could lead to engineered expression hosts with enhanced genome stability.

• Drug development platforms: The parallel between yeast HAM1 and human ITPA suggests that yeast systems could be developed as screening platforms for drugs targeting ITPA-related human diseases.

Researchers exploring these applications should consider:

These applications represent promising avenues for translating basic HAM1 research into practical biotechnology solutions.